Aug 26 2011
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Transcript of Aug 26 2011
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BT-202Netaji Subhas Institute of Technology,
Dwarka, New Delhi.Dr. Amita Pandey
Aug 26, 2011
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Learning check!
Serine has pK values of 2.19 and 9.21. What is its estimated pI?•2.19•5.70•7.00•9.21
By convention amino terminal residue of a peptide chain is written as the right-most residue.•True•False
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• The alpha amino groups of all amino acids have a charge at pH 7.0.positive
Which ionic species is most prevalent at the pI.• H2NCH2COOH
• H3N+CH2COOH
• H3N+CH2COO-
• H2NCH2COO-
In which pH range is glycine fully protonated.• Above pH 10.0• pH 2.35 - pH 9.78• pH 2.35 +1/-1• Below pH 1.0
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Which side chain is most likely to be negatively charged @ pH 7.0?•Arginine•Aspartic acid•Tyrosine•Cysteine
Which amino acid side chain is NOT aromatic?•Trytophan•Methionine•Tyrosine•Phenylalanine
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Structure of proteins
Proteins perform diverse functions in the cell.
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Primary Structure
Function of a protein depends upon its AA sequence.-proteins with different functions have different AA sequence.-various genetic disorders have defective proteins.-functionally related proteins have similar AA sequence.
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Polymorphic proteins
Same protein with different amino acid composition is called polymorphic protein.
-20%-30% proteins in humans are polymorphic. Eg ABO blood group.
-polymorphism also exists among distantly related species.
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First protein Sequence
Frederick Sanger (1953)AA sequence of hormoneinsulin
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End Group Analysis• Identify number of terminal AAs– Number of chains/subunits
• Identify specific AA• Sanger method (FDNB)
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End Group AnalysisDansyl chloride
•Reacts with primary amines
– N-terminus
•Yields dansylated polypeptides
•Dansylated polypeptides hydrolyzed to liberate the modified dansyl AA
•Dansyl AA can be identified by chromatography or spectroscopy (yellow fluorescence)
Dabsyl chloride
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Edman degradation
• Only single AA from the N-terminus is cleaved
• Entire protein sequence can be deduced• Identify using NMR, HPLC, etc.• Sequenator (entire process for proteins <
100 residues)
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Sequencing larger proteins
• Formation of smaller segments to assist with sequencing
• Process:– Cleave protein into specific fragments
• Chemically or enzymatically
• Break disulfide bonds
– Purify fragments
– Sequence fragments
– Determine order of fragments and disulfide bonds
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Breaking disulfide bond
• performic acid
• Cys residues form
cysteic acid
• Acid can oxidize
other residues, so
not ideal
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Breaking disulfide bond
β-2-Mercaptoethanol
(HSCH2CH2OH)
Dithiothreitol (DTT)
(HSCH2CH(OH)CH(OH)CH
2SH)
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Enzymatic and Chemical Cleavage
• Enzymatic
–Endopeptidases
• Chemical
-Cyanogen bromide (CNBr)
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peptide fragments
Sequencing
Edman procedure
Ordering of peptides
Cleaved again withdifferent enzyme or chemical
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Learning check
• A protein is cleaved with cyanogen bromide to yield the following sequences:– Arg-Ala-Tyr-Gly-Asn– Leu-Phe-Met– Asp-Met
• The same protein is cleaved with chymotrypsin to yield the following sequences: – Met-Arg-Ala-Tyr– Asp-Met-Leu-Phe– Gly-Asn
• What is the sequence of the protein?Asp-Met-Leu-Phe-Met-Arg-Ala-Tyr
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Protein sequencing
• Mass spectrometry-20-30 AA long fragments.
• Determination of protein sequence from DNA sequence-www.ncbi.nlm.nih.gov
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Genome sequencing
organism Size of genome Year
Haemophilus 1.8 Mb 1995
Saccharomyces 12.1 Mb 1996
C. elegans 100Mb 1998
Fruit fly 139 Mb 2005
Human 3.5 Gb 2006
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Consensus sequences
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Synthesis of peptides
Three ways to obtain a peptide are
-from the tissue-genetic engineering-chemical synthesis
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Chemical synthesis
9-fluorenylmethoxycarbonyl
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Proteins
• Make up about 15% of the cell• Have many functions in the
cell– Enzymes– Structural– Transport– Motor– Storage– Signaling– Receptors– Gene regulation
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Protein folding
• The peptide bond allows for rotation around it and therefore the protein can fold and orient the R groups in favorable positions
• Weak non-covalent interactions will hold the protein in its functional shape – these are weak and will take many to hold the shape
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Non-covalent bonds in protein folding
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Globular protein
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Hydrogen Bonds in Proteins
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Protein folding
• Proteins shape is determined by the sequence of the amino acids
• The final shape is called the conformation and has the lowest free energy possible
• Denaturation is the process of unfolding the protein– with heat, pH or chemical compounds
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Protein folding
• Renaturation is the process of protein regaining its native conformation
• Eg. Ribonuclease
• Molecular chaperones are small proteins that help guide the folding and can help keep the new protein from associating with the wrong partner
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Protein folding
-helix – protein turns like a spiral – fibrous proteins (hair, nails, horns)
-sheet – protein folds back on itself as in a ribbon –globularprotein
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Sheets
• Core of many proteins is the sheet
• Form rigid structures with the H-bond
• Can be of 2 types– Anti-parallel – run in
an opposite direction of its neighbor (A)
– Parallel – run in the same direction with longer looping sections between them (B)
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Helix
• Formed by a H-bond between every 4th peptide bond – C=O to N-H
• Usually in proteins that span a membrane
• The helix can either coil to the right or the left
• Can also coil around each other – coiled-coil shape
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Protein structure
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Domains
• A domaindomain is a basic structural unit of a protein structure – distinct from those that make up the conformations
• Part of protein that can fold into a stable structure independently
• Different domains can impart different functions to proteins
• Proteins can have one to many domains depending on protein size
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Domains
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Protein Familie
s
• Have similarities in amino acid sequence and 3-D structure
• Have similar functions such as breakdown proteins but do it differently
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Proteins – Multiple Peptides
• Non-covalent bonds can form interactions between individual polypeptide chains– Binding site – where proteins interact
with one another– Subunit – each polypeptide chain of
large protein– Dimer – protein made of 2 subunits• Can be same subunit or different subunits
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Single Subunit Proteins
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Different Subunit Proteins
• Hemoglobin–2 globin subunits–2 globin subunits
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Protein Assemblies
• Proteins can form very large assemblies
• Can form long chains if the protein has 2 binding sites – link together as a helix or a ring
• Actin fibers in muscles and cytoskeleton – is made from thousands of actin molecules as a helical fiber
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Types of Proteins
• Globular ProteinsGlobular Proteins – most of what we have dealt with so far– Compact shape like a ball with
irregular surfaces– Enzymes are globular
• Fibrous ProteinsFibrous Proteins – usually span a long distance in the cell– 3-D structure is usually long and rod
shaped
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Important Fibrous Proteins
• Intermediate filaments of the cytoskeleton – Structural scaffold inside the cell• Keratin in hair, horns and nails
• Extracellular matrix – Bind cells together to make tissues– Secreted from cells and assemble in
long fibers • Collagen – fiber with a glycine every third
amino acid in the protein• Elastin – unstructured fibers that gives
tissue an elastic characteristic
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Collagen and Elastin
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Stabilizing Cross-Links
• Cross linkages can be between 2 parts of a protein or between 2 subunits
• Disulfide bonds (S-S) form between adjacent -SH groups on the amino acid cysteine
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Proteins at Work
• The conformation of a protein gives it a unique function
• Ligand – the molecule that a protein can bind
• Binding site – part of the protein that interacts with the ligand– Consists of a cavity formed by a specific
arrangement of amino acids
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Ligand Binding
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Formation of Binding Site
• The binding site forms when amino acids from within the protein come together in the folding
• The remaining sequences may play a role in regulating the protein’s activity
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Antibody Family
• A family of proteins that can be created to bind to almost any molecule
• AntibodiesAntibodies (immunoglobulins) are made in response to a foreign molecule ie. bacteria, virus, pollen… called the antigenantigen
• Bind together tightly and therefore inactivates the antigen or marks it for destruction
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Antibodies
• Y-shaped molecules with 2 binding sites at the upper ends of the Y
• The loops of polypeptides on the end of the binding site are what imparts the recognition of the antigen
• Changes in the sequence of the loops make the antibody recognize different antigens - specificity
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Antibodies
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Binding Strength• Can be measured directly• Antibodies and antigens are mixing
around in a solution, eventually they will bump into each other in a way that the antigen sticks to the antibody, eventually they will separate due to the motion in the molecules
• This process continues until the equilibrium equilibrium is reached – number sticking is constant and number leaving is constant
• This can be determined for any protein and its ligandligand
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Equilibrium
Constant
• Concentration of antigen, antibody and antigen/antibody complex at equilibrium can be measured – equilibrium constant (K)equilibrium constant (K)
• Larger the K the tighter the binding or the more non-covalent bonds that hold the 2 together
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Enzymes as Catalysts
• Enzymes are proteins that bind to their ligand as the 1st step in a process
• An enzyme’s ligand is called a substratesubstrate– May be 1 or more molecules
• Output of the reaction is called the product
• Enzymes can repeat these steps many times and rapidly, called catalysts
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Enzymes at Work• Lysozyme is an important enzyme that
protects us from bacteria by making holes in the bacterial cell wall and causing it to break
• Lysozyme adds H2O to the glycosidic bond in the cell wall
• Lysozyme holds the polysaccharide in a position that allows the H2O to break the bond – this is the transition state transition state – state between substrate and product
• Active siteActive site is a special binding site in enzymes where the chemical reaction takes place
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Lysozyme
• Non-covalent bonds hold the polysaccharide in the active site until the reaction occurs
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Features of Enzyme Catalysis
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