Paper by Ramon K Tabtiang Brent O Cezairliyan Robert A Grant Jesse C Cochrane and Robert T Sauer - Department of Biology Massachusetts Institute of Technology Cambridge MA 02139

Presented by Gayathri Priya Ravichandran

Consolidating critical binding

determinants

by noncyclic rearrangement of protein

secondary structure

Structure

bull Lot of basicsbull Objectivebull Introductionbull Methodsbull Some discussionsbull Referencebull Questions

Basics ndash Levels of protein structurebull Secondary Structure The secondary structure of a

protein describes certain repetitive local conformations that are found in most peptide chains The secondary structure does not describe the actual folding the protein in three dimensions but instead illustrates the structure of small regions of the peptide

bull Primary Structure The primary structure of a biological molecule is the exact specification of its atomic composition and the chemical bonds connecting those atoms

Levels of protein structure

1 Primary ndash amino acid sequence and disulfide bonds2 Secondary ndash Local folding to form regular elements3 Tertiary ndash 3 Dimensional folded structure4 Quaternary ndash Tertiary structure of multimeric proteins

Alpha Helix

bull It is a secondary structure of proteins and is a righthand-coiled or spiral conformation (helix) in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier ( hydrogen bonding)

Structure

bull Lot of basicsbull Objectivebull Introductionbull Methodsbull Some discussionsbull Referencebull Questions

Basics ndash Levels of protein structurebull Secondary Structure The secondary structure of a

protein describes certain repetitive local conformations that are found in most peptide chains The secondary structure does not describe the actual folding the protein in three dimensions but instead illustrates the structure of small regions of the peptide

bull Primary Structure The primary structure of a biological molecule is the exact specification of its atomic composition and the chemical bonds connecting those atoms

Levels of protein structure

1 Primary ndash amino acid sequence and disulfide bonds2 Secondary ndash Local folding to form regular elements3 Tertiary ndash 3 Dimensional folded structure4 Quaternary ndash Tertiary structure of multimeric proteins

Alpha Helix

bull It is a secondary structure of proteins and is a righthand-coiled or spiral conformation (helix) in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier ( hydrogen bonding)

Basics ndash Levels of protein structurebull Secondary Structure The secondary structure of a

protein describes certain repetitive local conformations that are found in most peptide chains The secondary structure does not describe the actual folding the protein in three dimensions but instead illustrates the structure of small regions of the peptide

bull Primary Structure The primary structure of a biological molecule is the exact specification of its atomic composition and the chemical bonds connecting those atoms

Levels of protein structure

1 Primary ndash amino acid sequence and disulfide bonds2 Secondary ndash Local folding to form regular elements3 Tertiary ndash 3 Dimensional folded structure4 Quaternary ndash Tertiary structure of multimeric proteins

Alpha Helix

bull It is a secondary structure of proteins and is a righthand-coiled or spiral conformation (helix) in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier ( hydrogen bonding)

Levels of protein structure

1 Primary ndash amino acid sequence and disulfide bonds2 Secondary ndash Local folding to form regular elements3 Tertiary ndash 3 Dimensional folded structure4 Quaternary ndash Tertiary structure of multimeric proteins

Alpha Helix

bull It is a secondary structure of proteins and is a righthand-coiled or spiral conformation (helix) in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier ( hydrogen bonding)

Alpha Helix

bull It is a secondary structure of proteins and is a righthand-coiled or spiral conformation (helix) in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier ( hydrogen bonding)

Beta Sheet

bull The β sheet (also β-pleated sheet) is the second form of regular secondary structure in proteins bull Beta sheets consist of beta strands connected

laterally by at least two or three backbone hydrogen bonds forming a generally twisted pleated sheetbull A beta strand (also β strand) is a stretch of

polypeptide chain typically 3 to 10 amino acids long with backbone in an extended conformation

Beta sheet

Operatorbull In genetics an operator is a segment of DNA to which a

transcription factor binds to regulate gene expression The transcription factor is typically a repressor which can bind to the operator to prevent transcription

bull Transcription is the first step of gene expression in which a particular segment of DNA is copied into RNA (mRNA tRNA or rRNA) by the enzyme RNA polymerase

Repressorbull A repressor is a DNA- or RNA-binding protein that inhibits the

expression of one or more genes by binding to the operator or associated silencers

Transcription

Arc molecule

bull Arc is a member of the immediate-early gene (IEG) familybull It is a rapidly activated class of genes functionally

defined by their ability to be transcribed in the presence of protein synthesis inhibitors

Homodimerbull A protein composed of two identical polypeptide chainsbull Polypeptides are chains of amino acidsbull Peptides are naturally occurring biological molecules They are

short chains of amino acid monomers linked by peptide (amide) bonds

bull A peptide bond (amide bond) is a covalent chemical bond formed between two amino acid molecules

Objective

bull Design a single-chain variant of the Arc repressor homodimer bull Here1113088 strands that contact operator DNA are

connected by a hairpin turnbull The1113088 helices that form the tetrahelical scaffold of

the dimer are attached by a short linkerbull The designed protein represents a noncyclic

permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc) in which the two subunits are fused by a single linker which is very stable

Arc repressor binding to DNA

Beta sheets in contact with operator DNA

Tetrahelical bundle

Objectivebull The crystal structure of the permuted protein reveals an

essentially wild-type fold demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure

bull Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature

bull A phenotype is the composite of an organisms observable characteristics or traits such as its morphology development biochemical or physiological properties phenology behavior and products of behavior (such as a birds nest)

Introduction

bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact

bull Hydrophobic ndash water ldquofearingrdquo

Introduction

bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding

bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)

Methods

bull Molecular Biologybull Constructed in a plasmid system by using a

combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was

verified by DNA sequencing

PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies

Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells

were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by

absorbance using an extinction coefficientbull Protein purification is a series of processes intended to

isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms

Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc

were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get

isomorphous cell of the native crystal

Isomorphous ndash having same crystalline form

Result - Structure

Resultsbull Design As shown in Fig 1C the order of structural elements

in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold

Results

bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification

Results

bull Stability and Folding Kinetics bull Rearranging the order of secondary structural

elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation

Results - Analytical Ultracentrifuge

bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force

bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein

Results - Denaturation

bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar

Discussionbull Proteins are generally remarkably robust to amino acid

substitutions cyclic permutations insertions and even small deletions

bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate

bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations

Discussionbull The Arc fold was maintained after noncyclic rearrangement of

its secondary structural elements which required the addition of two non-natural linkers

bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc

Discussionbull Previous studies did not find noncyclic permutants of barnase

that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding

bull The protein design success mainly depends on the fold of the protein the topology and the required linkers

Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C

Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309

bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596

bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637

bull Wikipedia

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31

Beta sheet

Operatorbull In genetics an operator is a segment of DNA to which a

transcription factor binds to regulate gene expression The transcription factor is typically a repressor which can bind to the operator to prevent transcription

bull Transcription is the first step of gene expression in which a particular segment of DNA is copied into RNA (mRNA tRNA or rRNA) by the enzyme RNA polymerase

Repressorbull A repressor is a DNA- or RNA-binding protein that inhibits the

expression of one or more genes by binding to the operator or associated silencers

Transcription

Arc molecule

bull Arc is a member of the immediate-early gene (IEG) familybull It is a rapidly activated class of genes functionally

defined by their ability to be transcribed in the presence of protein synthesis inhibitors

Homodimerbull A protein composed of two identical polypeptide chainsbull Polypeptides are chains of amino acidsbull Peptides are naturally occurring biological molecules They are

short chains of amino acid monomers linked by peptide (amide) bonds

bull A peptide bond (amide bond) is a covalent chemical bond formed between two amino acid molecules

Objective

bull Design a single-chain variant of the Arc repressor homodimer bull Here1113088 strands that contact operator DNA are

connected by a hairpin turnbull The1113088 helices that form the tetrahelical scaffold of

the dimer are attached by a short linkerbull The designed protein represents a noncyclic

permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc) in which the two subunits are fused by a single linker which is very stable

Arc repressor binding to DNA

Beta sheets in contact with operator DNA

Tetrahelical bundle

Objectivebull The crystal structure of the permuted protein reveals an

essentially wild-type fold demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure

bull Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature

bull A phenotype is the composite of an organisms observable characteristics or traits such as its morphology development biochemical or physiological properties phenology behavior and products of behavior (such as a birds nest)

Introduction

bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact

bull Hydrophobic ndash water ldquofearingrdquo

Introduction

bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding

bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)

Methods

bull Molecular Biologybull Constructed in a plasmid system by using a

combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was

verified by DNA sequencing

PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies

Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells

were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by

absorbance using an extinction coefficientbull Protein purification is a series of processes intended to

isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms

Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc

were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get

isomorphous cell of the native crystal

Isomorphous ndash having same crystalline form

Result - Structure

Resultsbull Design As shown in Fig 1C the order of structural elements

in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold

Results

bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification

Results

bull Stability and Folding Kinetics bull Rearranging the order of secondary structural

elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation

Results - Analytical Ultracentrifuge

bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force

bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein

Results - Denaturation

bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar

Discussionbull Proteins are generally remarkably robust to amino acid

substitutions cyclic permutations insertions and even small deletions

bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate

bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations

Discussionbull The Arc fold was maintained after noncyclic rearrangement of

its secondary structural elements which required the addition of two non-natural linkers

bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc

Discussionbull Previous studies did not find noncyclic permutants of barnase

that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding

bull The protein design success mainly depends on the fold of the protein the topology and the required linkers

Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C

Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309

bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596

bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637

bull Wikipedia

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31

Operatorbull In genetics an operator is a segment of DNA to which a

transcription factor binds to regulate gene expression The transcription factor is typically a repressor which can bind to the operator to prevent transcription

bull Transcription is the first step of gene expression in which a particular segment of DNA is copied into RNA (mRNA tRNA or rRNA) by the enzyme RNA polymerase

Repressorbull A repressor is a DNA- or RNA-binding protein that inhibits the

expression of one or more genes by binding to the operator or associated silencers

Transcription

Arc molecule

bull Arc is a member of the immediate-early gene (IEG) familybull It is a rapidly activated class of genes functionally

defined by their ability to be transcribed in the presence of protein synthesis inhibitors

Homodimerbull A protein composed of two identical polypeptide chainsbull Polypeptides are chains of amino acidsbull Peptides are naturally occurring biological molecules They are

short chains of amino acid monomers linked by peptide (amide) bonds

bull A peptide bond (amide bond) is a covalent chemical bond formed between two amino acid molecules

Objective

bull Design a single-chain variant of the Arc repressor homodimer bull Here1113088 strands that contact operator DNA are

connected by a hairpin turnbull The1113088 helices that form the tetrahelical scaffold of

the dimer are attached by a short linkerbull The designed protein represents a noncyclic

permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc) in which the two subunits are fused by a single linker which is very stable

Arc repressor binding to DNA

Beta sheets in contact with operator DNA

Tetrahelical bundle

Objectivebull The crystal structure of the permuted protein reveals an

essentially wild-type fold demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure

bull Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature

bull A phenotype is the composite of an organisms observable characteristics or traits such as its morphology development biochemical or physiological properties phenology behavior and products of behavior (such as a birds nest)

Introduction

bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact

bull Hydrophobic ndash water ldquofearingrdquo

Introduction

bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding

bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)

Methods

bull Molecular Biologybull Constructed in a plasmid system by using a

combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was

verified by DNA sequencing

PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies

Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells

were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by

absorbance using an extinction coefficientbull Protein purification is a series of processes intended to

isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms

Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc

were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get

isomorphous cell of the native crystal

Isomorphous ndash having same crystalline form

Result - Structure

Resultsbull Design As shown in Fig 1C the order of structural elements

in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold

Results

bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification

Results

bull Stability and Folding Kinetics bull Rearranging the order of secondary structural

elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation

Results - Analytical Ultracentrifuge

bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force

bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein

Results - Denaturation

bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar

Discussionbull Proteins are generally remarkably robust to amino acid

substitutions cyclic permutations insertions and even small deletions

bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate

bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations

Discussionbull The Arc fold was maintained after noncyclic rearrangement of

its secondary structural elements which required the addition of two non-natural linkers

bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc

Discussionbull Previous studies did not find noncyclic permutants of barnase

that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding

bull The protein design success mainly depends on the fold of the protein the topology and the required linkers

Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C

Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309

bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596

bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637

bull Wikipedia

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31

Repressorbull A repressor is a DNA- or RNA-binding protein that inhibits the

expression of one or more genes by binding to the operator or associated silencers

Transcription

Arc molecule

bull Arc is a member of the immediate-early gene (IEG) familybull It is a rapidly activated class of genes functionally

defined by their ability to be transcribed in the presence of protein synthesis inhibitors

Homodimerbull A protein composed of two identical polypeptide chainsbull Polypeptides are chains of amino acidsbull Peptides are naturally occurring biological molecules They are

short chains of amino acid monomers linked by peptide (amide) bonds

bull A peptide bond (amide bond) is a covalent chemical bond formed between two amino acid molecules

Objective

bull Design a single-chain variant of the Arc repressor homodimer bull Here1113088 strands that contact operator DNA are

connected by a hairpin turnbull The1113088 helices that form the tetrahelical scaffold of

the dimer are attached by a short linkerbull The designed protein represents a noncyclic

permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc) in which the two subunits are fused by a single linker which is very stable

Arc repressor binding to DNA

Beta sheets in contact with operator DNA

Tetrahelical bundle

Objectivebull The crystal structure of the permuted protein reveals an

essentially wild-type fold demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure

bull Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature

bull A phenotype is the composite of an organisms observable characteristics or traits such as its morphology development biochemical or physiological properties phenology behavior and products of behavior (such as a birds nest)

Introduction

bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact

bull Hydrophobic ndash water ldquofearingrdquo

Introduction

bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding

bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)

Methods

bull Molecular Biologybull Constructed in a plasmid system by using a

combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was

verified by DNA sequencing

PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies

Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells

were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by

absorbance using an extinction coefficientbull Protein purification is a series of processes intended to

isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms

Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc

were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get

isomorphous cell of the native crystal

Isomorphous ndash having same crystalline form

Result - Structure

Resultsbull Design As shown in Fig 1C the order of structural elements

in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold

Results

bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification

Results

bull Stability and Folding Kinetics bull Rearranging the order of secondary structural

elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation

Results - Analytical Ultracentrifuge

bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force

bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein

Results - Denaturation

bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar

Discussionbull Proteins are generally remarkably robust to amino acid

substitutions cyclic permutations insertions and even small deletions

bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate

bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations

Discussionbull The Arc fold was maintained after noncyclic rearrangement of

its secondary structural elements which required the addition of two non-natural linkers

bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc

Discussionbull Previous studies did not find noncyclic permutants of barnase

that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding

bull The protein design success mainly depends on the fold of the protein the topology and the required linkers

Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C

Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309

bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596

bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637

bull Wikipedia

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31

Transcription

Arc molecule

bull Arc is a member of the immediate-early gene (IEG) familybull It is a rapidly activated class of genes functionally

defined by their ability to be transcribed in the presence of protein synthesis inhibitors

Homodimerbull A protein composed of two identical polypeptide chainsbull Polypeptides are chains of amino acidsbull Peptides are naturally occurring biological molecules They are

short chains of amino acid monomers linked by peptide (amide) bonds

bull A peptide bond (amide bond) is a covalent chemical bond formed between two amino acid molecules

Objective

bull Design a single-chain variant of the Arc repressor homodimer bull Here1113088 strands that contact operator DNA are

connected by a hairpin turnbull The1113088 helices that form the tetrahelical scaffold of

the dimer are attached by a short linkerbull The designed protein represents a noncyclic

permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc) in which the two subunits are fused by a single linker which is very stable

Arc repressor binding to DNA

Beta sheets in contact with operator DNA

Tetrahelical bundle

Objectivebull The crystal structure of the permuted protein reveals an

essentially wild-type fold demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure

bull Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature

bull A phenotype is the composite of an organisms observable characteristics or traits such as its morphology development biochemical or physiological properties phenology behavior and products of behavior (such as a birds nest)

Introduction

bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact

bull Hydrophobic ndash water ldquofearingrdquo

Introduction

bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding

bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)

Methods

bull Molecular Biologybull Constructed in a plasmid system by using a

combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was

verified by DNA sequencing

PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies

Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells

were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by

absorbance using an extinction coefficientbull Protein purification is a series of processes intended to

isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms

Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc

were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get

isomorphous cell of the native crystal

Isomorphous ndash having same crystalline form

Result - Structure

Resultsbull Design As shown in Fig 1C the order of structural elements

in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold

Results

bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification

Results

bull Stability and Folding Kinetics bull Rearranging the order of secondary structural

elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation

Results - Analytical Ultracentrifuge

bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force

bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein

Results - Denaturation

bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar

Discussionbull Proteins are generally remarkably robust to amino acid

substitutions cyclic permutations insertions and even small deletions

bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate

bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations

Discussionbull The Arc fold was maintained after noncyclic rearrangement of

its secondary structural elements which required the addition of two non-natural linkers

bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc

Discussionbull Previous studies did not find noncyclic permutants of barnase

that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding

bull The protein design success mainly depends on the fold of the protein the topology and the required linkers

Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C

Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309

bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596

bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637

bull Wikipedia

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31

Arc molecule

bull Arc is a member of the immediate-early gene (IEG) familybull It is a rapidly activated class of genes functionally

defined by their ability to be transcribed in the presence of protein synthesis inhibitors

Homodimerbull A protein composed of two identical polypeptide chainsbull Polypeptides are chains of amino acidsbull Peptides are naturally occurring biological molecules They are

short chains of amino acid monomers linked by peptide (amide) bonds

bull A peptide bond (amide bond) is a covalent chemical bond formed between two amino acid molecules

Objective

bull Design a single-chain variant of the Arc repressor homodimer bull Here1113088 strands that contact operator DNA are

connected by a hairpin turnbull The1113088 helices that form the tetrahelical scaffold of

the dimer are attached by a short linkerbull The designed protein represents a noncyclic

permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc) in which the two subunits are fused by a single linker which is very stable

Arc repressor binding to DNA

Beta sheets in contact with operator DNA

Tetrahelical bundle

Objectivebull The crystal structure of the permuted protein reveals an

essentially wild-type fold demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure

bull Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature

bull A phenotype is the composite of an organisms observable characteristics or traits such as its morphology development biochemical or physiological properties phenology behavior and products of behavior (such as a birds nest)

Introduction

bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact

bull Hydrophobic ndash water ldquofearingrdquo

Introduction

bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding

bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)

Methods

bull Molecular Biologybull Constructed in a plasmid system by using a

combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was

verified by DNA sequencing

PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies

Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells

were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by

absorbance using an extinction coefficientbull Protein purification is a series of processes intended to

isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms

Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc

were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get

isomorphous cell of the native crystal

Isomorphous ndash having same crystalline form

Result - Structure

Resultsbull Design As shown in Fig 1C the order of structural elements

in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold

Results

bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification

Results

bull Stability and Folding Kinetics bull Rearranging the order of secondary structural

elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation

Results - Analytical Ultracentrifuge

bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force

bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein

Results - Denaturation

bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar

Discussionbull Proteins are generally remarkably robust to amino acid

substitutions cyclic permutations insertions and even small deletions

bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate

bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations

Discussionbull The Arc fold was maintained after noncyclic rearrangement of

its secondary structural elements which required the addition of two non-natural linkers

bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc

Discussionbull Previous studies did not find noncyclic permutants of barnase

that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding

bull The protein design success mainly depends on the fold of the protein the topology and the required linkers

Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C

Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309

bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596

bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637

bull Wikipedia

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31

Homodimerbull A protein composed of two identical polypeptide chainsbull Polypeptides are chains of amino acidsbull Peptides are naturally occurring biological molecules They are

short chains of amino acid monomers linked by peptide (amide) bonds

bull A peptide bond (amide bond) is a covalent chemical bond formed between two amino acid molecules

Objective

bull Design a single-chain variant of the Arc repressor homodimer bull Here1113088 strands that contact operator DNA are

connected by a hairpin turnbull The1113088 helices that form the tetrahelical scaffold of

the dimer are attached by a short linkerbull The designed protein represents a noncyclic

permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc) in which the two subunits are fused by a single linker which is very stable

Arc repressor binding to DNA

Beta sheets in contact with operator DNA

Tetrahelical bundle

Objectivebull The crystal structure of the permuted protein reveals an

essentially wild-type fold demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure

bull Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature

bull A phenotype is the composite of an organisms observable characteristics or traits such as its morphology development biochemical or physiological properties phenology behavior and products of behavior (such as a birds nest)

Introduction

bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact

bull Hydrophobic ndash water ldquofearingrdquo

Introduction

bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding

bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)

Methods

bull Molecular Biologybull Constructed in a plasmid system by using a

combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was

verified by DNA sequencing

PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies

Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells

were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by

absorbance using an extinction coefficientbull Protein purification is a series of processes intended to

isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms

Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc

were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get

isomorphous cell of the native crystal

Isomorphous ndash having same crystalline form

Result - Structure

Resultsbull Design As shown in Fig 1C the order of structural elements

in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold

Results

bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification

Results

bull Stability and Folding Kinetics bull Rearranging the order of secondary structural

elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation

Results - Analytical Ultracentrifuge

bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force

bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein

Results - Denaturation

bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar

Discussionbull Proteins are generally remarkably robust to amino acid

substitutions cyclic permutations insertions and even small deletions

bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate

bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations

Discussionbull The Arc fold was maintained after noncyclic rearrangement of

its secondary structural elements which required the addition of two non-natural linkers

bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc

Discussionbull Previous studies did not find noncyclic permutants of barnase

that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding

bull The protein design success mainly depends on the fold of the protein the topology and the required linkers

Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C

Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309

bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596

bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637

bull Wikipedia

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31

Objective

bull Design a single-chain variant of the Arc repressor homodimer bull Here1113088 strands that contact operator DNA are

connected by a hairpin turnbull The1113088 helices that form the tetrahelical scaffold of

the dimer are attached by a short linkerbull The designed protein represents a noncyclic

permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc) in which the two subunits are fused by a single linker which is very stable

Arc repressor binding to DNA

Beta sheets in contact with operator DNA

Tetrahelical bundle

Objectivebull The crystal structure of the permuted protein reveals an

essentially wild-type fold demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure

bull Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature

bull A phenotype is the composite of an organisms observable characteristics or traits such as its morphology development biochemical or physiological properties phenology behavior and products of behavior (such as a birds nest)

Introduction

bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact

bull Hydrophobic ndash water ldquofearingrdquo

Introduction

bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding

bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)

Methods

bull Molecular Biologybull Constructed in a plasmid system by using a

combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was

verified by DNA sequencing

PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies

Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells

were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by

absorbance using an extinction coefficientbull Protein purification is a series of processes intended to

isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms

Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc

were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get

isomorphous cell of the native crystal

Isomorphous ndash having same crystalline form

Result - Structure

Resultsbull Design As shown in Fig 1C the order of structural elements

in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold

Results

bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification

Results

bull Stability and Folding Kinetics bull Rearranging the order of secondary structural

elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation

Results - Analytical Ultracentrifuge

bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force

bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein

Results - Denaturation

bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar

Discussionbull Proteins are generally remarkably robust to amino acid

substitutions cyclic permutations insertions and even small deletions

bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate

bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations

Discussionbull The Arc fold was maintained after noncyclic rearrangement of

its secondary structural elements which required the addition of two non-natural linkers

bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc

Discussionbull Previous studies did not find noncyclic permutants of barnase

that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding

bull The protein design success mainly depends on the fold of the protein the topology and the required linkers

Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C

Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309

bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596

bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637

bull Wikipedia

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31

Arc repressor binding to DNA

Beta sheets in contact with operator DNA

Tetrahelical bundle

Objectivebull The crystal structure of the permuted protein reveals an

essentially wild-type fold demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure

bull Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature

bull A phenotype is the composite of an organisms observable characteristics or traits such as its morphology development biochemical or physiological properties phenology behavior and products of behavior (such as a birds nest)

Introduction

bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact

bull Hydrophobic ndash water ldquofearingrdquo

Introduction

bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding

bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)

Methods

bull Molecular Biologybull Constructed in a plasmid system by using a

combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was

verified by DNA sequencing

PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies

Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells

were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by

absorbance using an extinction coefficientbull Protein purification is a series of processes intended to

isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms

Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc

were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get

isomorphous cell of the native crystal

Isomorphous ndash having same crystalline form

Result - Structure

Resultsbull Design As shown in Fig 1C the order of structural elements

in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold

Results

bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification

Results

bull Stability and Folding Kinetics bull Rearranging the order of secondary structural

elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation

Results - Analytical Ultracentrifuge

bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force

bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein

Results - Denaturation

bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar

Discussionbull Proteins are generally remarkably robust to amino acid

substitutions cyclic permutations insertions and even small deletions

bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate

bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations

Discussionbull The Arc fold was maintained after noncyclic rearrangement of

its secondary structural elements which required the addition of two non-natural linkers

bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc

Discussionbull Previous studies did not find noncyclic permutants of barnase

that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding

bull The protein design success mainly depends on the fold of the protein the topology and the required linkers

Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C

Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309

bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596

bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637

bull Wikipedia

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31

Objectivebull The crystal structure of the permuted protein reveals an

essentially wild-type fold demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure

bull Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature

bull A phenotype is the composite of an organisms observable characteristics or traits such as its morphology development biochemical or physiological properties phenology behavior and products of behavior (such as a birds nest)

Introduction

bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact

bull Hydrophobic ndash water ldquofearingrdquo

Introduction

bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding

bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)

Methods

bull Molecular Biologybull Constructed in a plasmid system by using a

combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was

verified by DNA sequencing

PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies

Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells

were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by

absorbance using an extinction coefficientbull Protein purification is a series of processes intended to

isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms

Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc

were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get

isomorphous cell of the native crystal

Isomorphous ndash having same crystalline form

Result - Structure

Resultsbull Design As shown in Fig 1C the order of structural elements

in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold

Results

bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification

Results

bull Stability and Folding Kinetics bull Rearranging the order of secondary structural

elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation

Results - Analytical Ultracentrifuge

bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force

bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein

Results - Denaturation

bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar

Discussionbull Proteins are generally remarkably robust to amino acid

substitutions cyclic permutations insertions and even small deletions

bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate

bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations

Discussionbull The Arc fold was maintained after noncyclic rearrangement of

its secondary structural elements which required the addition of two non-natural linkers

bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc

Discussionbull Previous studies did not find noncyclic permutants of barnase

that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding

bull The protein design success mainly depends on the fold of the protein the topology and the required linkers

Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C

Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309

bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596

bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637

bull Wikipedia

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31

Introduction

bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact

bull Hydrophobic ndash water ldquofearingrdquo

Introduction

bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding

bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)

Methods

bull Molecular Biologybull Constructed in a plasmid system by using a

combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was

verified by DNA sequencing

PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies

Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells

were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by

absorbance using an extinction coefficientbull Protein purification is a series of processes intended to

isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms

Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc

were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get

isomorphous cell of the native crystal

Isomorphous ndash having same crystalline form

Result - Structure

Resultsbull Design As shown in Fig 1C the order of structural elements

in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold

Results

bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification

Results

bull Stability and Folding Kinetics bull Rearranging the order of secondary structural

elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation

Results - Analytical Ultracentrifuge

bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force

bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein

Results - Denaturation

bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar

Discussionbull Proteins are generally remarkably robust to amino acid

substitutions cyclic permutations insertions and even small deletions

bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate

bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations

Discussionbull The Arc fold was maintained after noncyclic rearrangement of

its secondary structural elements which required the addition of two non-natural linkers

bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc

Discussionbull Previous studies did not find noncyclic permutants of barnase

that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding

bull The protein design success mainly depends on the fold of the protein the topology and the required linkers

Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C

Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309

bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596

bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637

bull Wikipedia

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31

Introduction

bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding

bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)

Methods

bull Molecular Biologybull Constructed in a plasmid system by using a

combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was

verified by DNA sequencing

PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies

Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells

were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by

absorbance using an extinction coefficientbull Protein purification is a series of processes intended to

isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms

Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc

were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get

isomorphous cell of the native crystal

Isomorphous ndash having same crystalline form

Result - Structure

Resultsbull Design As shown in Fig 1C the order of structural elements

in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold

Results

bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification

Results

bull Stability and Folding Kinetics bull Rearranging the order of secondary structural

elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation

Results - Analytical Ultracentrifuge

bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force

bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein

Results - Denaturation

bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar

Discussionbull Proteins are generally remarkably robust to amino acid

substitutions cyclic permutations insertions and even small deletions

bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate

bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations

Discussionbull The Arc fold was maintained after noncyclic rearrangement of

its secondary structural elements which required the addition of two non-natural linkers

bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc

Discussionbull Previous studies did not find noncyclic permutants of barnase

that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding

bull The protein design success mainly depends on the fold of the protein the topology and the required linkers

Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C

Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309

bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596

bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637

bull Wikipedia

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31

Methods

bull Molecular Biologybull Constructed in a plasmid system by using a

combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was

verified by DNA sequencing

PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies

Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells

were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by

absorbance using an extinction coefficientbull Protein purification is a series of processes intended to

isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms

Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc

were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get

isomorphous cell of the native crystal

Isomorphous ndash having same crystalline form

Result - Structure

Resultsbull Design As shown in Fig 1C the order of structural elements

in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold

Results

bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification

Results

bull Stability and Folding Kinetics bull Rearranging the order of secondary structural

elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation

Results - Analytical Ultracentrifuge

bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force

bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein

Results - Denaturation

bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar

Discussionbull Proteins are generally remarkably robust to amino acid

substitutions cyclic permutations insertions and even small deletions

bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate

bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations

Discussionbull The Arc fold was maintained after noncyclic rearrangement of

its secondary structural elements which required the addition of two non-natural linkers

bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc

Discussionbull Previous studies did not find noncyclic permutants of barnase

that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding

bull The protein design success mainly depends on the fold of the protein the topology and the required linkers

Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C

Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309

bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596

bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637

bull Wikipedia

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31

Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells

were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by

absorbance using an extinction coefficientbull Protein purification is a series of processes intended to

isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms

Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc

were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get

isomorphous cell of the native crystal

Isomorphous ndash having same crystalline form

Result - Structure

Resultsbull Design As shown in Fig 1C the order of structural elements

in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold

Results

bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification

Results

bull Stability and Folding Kinetics bull Rearranging the order of secondary structural

elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation

Results - Analytical Ultracentrifuge

bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force

bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein

Results - Denaturation

bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar

Discussionbull Proteins are generally remarkably robust to amino acid

substitutions cyclic permutations insertions and even small deletions

bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate

bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations

Discussionbull The Arc fold was maintained after noncyclic rearrangement of

its secondary structural elements which required the addition of two non-natural linkers

bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc

Discussionbull Previous studies did not find noncyclic permutants of barnase

that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding

bull The protein design success mainly depends on the fold of the protein the topology and the required linkers

Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C

Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309

bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596

bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637

bull Wikipedia

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31

Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc

were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get

isomorphous cell of the native crystal

Isomorphous ndash having same crystalline form

Result - Structure

Resultsbull Design As shown in Fig 1C the order of structural elements

in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold

Results

bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification

Results

bull Stability and Folding Kinetics bull Rearranging the order of secondary structural

elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation

Results - Analytical Ultracentrifuge

bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force

bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein

Results - Denaturation

bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar

Discussionbull Proteins are generally remarkably robust to amino acid

substitutions cyclic permutations insertions and even small deletions

bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate

bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations

Discussionbull The Arc fold was maintained after noncyclic rearrangement of

its secondary structural elements which required the addition of two non-natural linkers

bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc

Discussionbull Previous studies did not find noncyclic permutants of barnase

that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding

bull The protein design success mainly depends on the fold of the protein the topology and the required linkers

Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C

Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309

bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596

bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637

bull Wikipedia

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31

Result - Structure

Resultsbull Design As shown in Fig 1C the order of structural elements

in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold

Results

bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification

Results

bull Stability and Folding Kinetics bull Rearranging the order of secondary structural

elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation

Results - Analytical Ultracentrifuge

bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force

bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein

Results - Denaturation

bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar

Discussionbull Proteins are generally remarkably robust to amino acid

substitutions cyclic permutations insertions and even small deletions

bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate

bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations

Discussionbull The Arc fold was maintained after noncyclic rearrangement of

its secondary structural elements which required the addition of two non-natural linkers

bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc

Discussionbull Previous studies did not find noncyclic permutants of barnase

that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding

bull The protein design success mainly depends on the fold of the protein the topology and the required linkers

Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C

Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309

bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596

bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637

bull Wikipedia

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31

Resultsbull Design As shown in Fig 1C the order of structural elements

in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold

Results

bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification

Results

bull Stability and Folding Kinetics bull Rearranging the order of secondary structural

elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation

Results - Analytical Ultracentrifuge

bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force

bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein

Results - Denaturation

bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar

Discussionbull Proteins are generally remarkably robust to amino acid

substitutions cyclic permutations insertions and even small deletions

bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate

bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations

Discussionbull The Arc fold was maintained after noncyclic rearrangement of

its secondary structural elements which required the addition of two non-natural linkers

bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc

Discussionbull Previous studies did not find noncyclic permutants of barnase

that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding

bull The protein design success mainly depends on the fold of the protein the topology and the required linkers

Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C

Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309

bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596

bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637

bull Wikipedia

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31

Results

bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification

Results

bull Stability and Folding Kinetics bull Rearranging the order of secondary structural

elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation

Results - Analytical Ultracentrifuge

bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force

bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein

Results - Denaturation

bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar

Discussionbull Proteins are generally remarkably robust to amino acid

substitutions cyclic permutations insertions and even small deletions

bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate

bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations

Discussionbull The Arc fold was maintained after noncyclic rearrangement of

its secondary structural elements which required the addition of two non-natural linkers

bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc

Discussionbull Previous studies did not find noncyclic permutants of barnase

that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding

bull The protein design success mainly depends on the fold of the protein the topology and the required linkers

Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C

Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309

bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596

bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637

bull Wikipedia

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31

Results

bull Stability and Folding Kinetics bull Rearranging the order of secondary structural

elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation

Results - Analytical Ultracentrifuge

bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force

bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein

Results - Denaturation

bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar

Discussionbull Proteins are generally remarkably robust to amino acid

substitutions cyclic permutations insertions and even small deletions

bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate

bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations

Discussionbull The Arc fold was maintained after noncyclic rearrangement of

its secondary structural elements which required the addition of two non-natural linkers

bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc

Discussionbull Previous studies did not find noncyclic permutants of barnase

that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding

bull The protein design success mainly depends on the fold of the protein the topology and the required linkers

Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C

Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309

bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596

bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637

bull Wikipedia

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31

Results - Analytical Ultracentrifuge

bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force

bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein

Results - Denaturation

bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar

Discussionbull Proteins are generally remarkably robust to amino acid

substitutions cyclic permutations insertions and even small deletions

bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate

bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations

Discussionbull The Arc fold was maintained after noncyclic rearrangement of

its secondary structural elements which required the addition of two non-natural linkers

bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc

Discussionbull Previous studies did not find noncyclic permutants of barnase

that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding

bull The protein design success mainly depends on the fold of the protein the topology and the required linkers

Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C

Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309

bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596

bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637

bull Wikipedia

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31

Results - Denaturation

bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar

Discussionbull Proteins are generally remarkably robust to amino acid

substitutions cyclic permutations insertions and even small deletions

bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate

bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations

Discussionbull The Arc fold was maintained after noncyclic rearrangement of

its secondary structural elements which required the addition of two non-natural linkers

bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc

Discussionbull Previous studies did not find noncyclic permutants of barnase

that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding

bull The protein design success mainly depends on the fold of the protein the topology and the required linkers

Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C

Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309

bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596

bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637

bull Wikipedia

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31

Discussionbull Proteins are generally remarkably robust to amino acid

substitutions cyclic permutations insertions and even small deletions

bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate

bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations

Discussionbull The Arc fold was maintained after noncyclic rearrangement of

its secondary structural elements which required the addition of two non-natural linkers

bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc

Discussionbull Previous studies did not find noncyclic permutants of barnase

that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding

bull The protein design success mainly depends on the fold of the protein the topology and the required linkers

Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C

Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309

bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596

bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637

bull Wikipedia

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31

Discussionbull The Arc fold was maintained after noncyclic rearrangement of

its secondary structural elements which required the addition of two non-natural linkers

bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc

Discussionbull Previous studies did not find noncyclic permutants of barnase

that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding

bull The protein design success mainly depends on the fold of the protein the topology and the required linkers

Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C

Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309

bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596

bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637

bull Wikipedia

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31

Discussionbull Previous studies did not find noncyclic permutants of barnase

that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding

bull The protein design success mainly depends on the fold of the protein the topology and the required linkers

Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C

Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309

bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596

bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637

bull Wikipedia

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31

Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C

Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309

bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596

bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637

bull Wikipedia

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31

Questions

• Slide 1
• Structure
• Basics ndash Levels of protein structure
• Levels of protein structure
• Alpha Helix
• Beta Sheet
• Beta sheet
• Operator
• Repressor
• Transcription
• Arc molecule
• Homodimer
• Objective
• Arc repressor binding to DNA
• Objective (2)
• Introduction
• Introduction (2)
• Methods
• Methods (2)
• Methods (3)
• Result - Structure
• Results
• Results (2)
• Results (3)
• Results - Analytical Ultracentrifuge
• Results - Denaturation
• Discussion
• Discussion (2)
• Discussion (3)
• Reference
• Slide 31