Chapter foundations of Chapter M A R K E T I N G Marketing Strategy and the Marketing Plan 5.
G & G Chapter 5
Transcript of G & G Chapter 5
CHAPTER 5Proteins: Their BiologicalFunctions and Primary
Structure
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5.1 Proteins are Linear Polymers of Amino AcidsProteins are linear polymers of amino acids:
The Peptide BondThe Peptide Bond• Bond occurs between the αα-amino group of one amino acid and the αα-carboxyl group of another amino acid
• A condensation reaction where the elements of H20 are removed
N N - C - COOH
H
H
HH
HHC - OHNH2 - C - C - OH
H
H OO
N N - C - COOH
H
H
HH
C NH2 - C - C
H
H OO
H H -OH- OH
C NH2 - C - C
H
H OO
N N - C - COOH
H
H
HH
The Peptide Bond!!The Peptide Bond!!
The Peptide Bond• is usually found in the trans conformation
• has partial (40%) double bond character
• is about 0.133 nm long - shorter than a typicalsingle bond but longer than a double bond
• Due to the double bond character, the sixatoms of the peptide bond group are alwaysplanar!
• N partially positive; O partially negative
THE PEPTIDE BOND:
C NH2 - C - C
H
H OO
N N - C - COOH
H
H
HH
The Peptide Bond is a Resonance Structure:
C NH2 - C - C
H
H O-O-
N+ N+ - C - COOH
H
H
HH
The Coplanar Nature of the Peptide Bond
Six atoms of the peptide group lie in a plane!
The coplanar nature of the Peptide Bond
“Peptides”
• Short polymers of amino acids
• Each unit is called a residue
• 2 residues - dipeptide
• 3 residues - tripeptide
• 12-20 residues - oligopeptide
• many - polypeptide
“Peptides”
“Protein”
One or more polypeptide chains
• One polypeptide chain - a monomeric protein
• More than one - multimeric protein
• Homomultimer - one kind of chain
• Heteromultimer - two or more different chains
• Hemoglobin, for example, is a heterotetramer
• It has two alpha chains and two beta chains
“Protein”
The tetrameric structure of hemoglobin
Proteins - Large and Small
• Insulin - A chain of 21 residues, B chain of 30residues -total mol. wt. of 5,733
• Glutamine synthetase - 12 subunits of 468residues each - total mol. wt. of 600,000
• Connectin proteins - alpha - MW 2.8 million!
• beta connectin - MW of 2.1 million, with alength of 1000 nm -it can stretch to 3000 nm!
Proteins - Large and Small
The Sequence of Amino Acids ina Protein
• is a unique characteristic of every protein
• is encoded by the nucleotide sequence ofDNA
• is thus a form of genetic information
• is read from the amino terminus to thecarboxyl terminus
The Sequence of Amino Acidsin a Protein:
• Protein chains have a direction.• Protein chains have a direction.
• By convention the N-terminus is taken to be the beginning of a polypeptide chain.
• By convention the N-terminus is taken to be the beginning of a polypeptide chain.
NH2 - C - C - N - C - C -N - C - COOHNH
2 - C - C - N - C - C -N - C - COOH
OO OOHH
HHHH HH
HH HH
HH CH3
CH3
Glycine-Glycine-AlanineGlycine-Glycine-Alanine
The sequence of ribonuclease A
5.2 Architecture of Proteins
• Shape - globular, fibrous, membrane
• The levels of protein structure
- Primary - linear amino acid sequence
- Secondary - peptide backbone - H-bonds
- Tertiary - overall 3-dimensional shape
- Quaternary - subunit organization
Architecture of Proteins
What forces determine thestructure?
• Primary structure - determined by covalent bonds
• Secondary, Tertiary, Quaternary structures - alldetermined by weak forces
• Weak forces - H-bonds, ionic interactions, vander Waals interactions, hydrophobic interactions
What forces determine the structure?
The tetrameric structure of hemoglobin
How to view a protein?
• backbone only
• backbone plus side chains
• ribbon structure
• space-filling structure
How to View a Protein?
Configuration andconformation arenot the same
5.3 Biological Functions ofProteinsProteins are the agents of biological function
• Enzymes - Ribonuclease
• Regulatory proteins - Insulin
• Transport proteins - Hemoglobin
• Structural proteins - Collagen
• Contractile proteins - Actin, Myosin
• Exotic proteins - Antifreeze proteins in fish
• Storage - seed storage proteins in plants
Biological Functions of Proteins:
5.4 Other Chemical Groups inProteins
Proteins may be "conjugated" with otherchemical groups
• If the non-amino acid part of the protein isimportant to its function, it is called aprosthetic group.
• Large organic molecules (vitamins)comjugated to proteins are coenzymes.
• Be familiar with the terms: glycoprotein,lipoprotein, nucleoprotein, phosphoprotein,metalloprotein, hemoprotein, flavoprotein.
Other Chemical Groups in Proteins:
5.7 Sequence DeterminationFrederick Sanger was the first - in 1953, he
sequenced the two chains of insulin.
• Sanger's results established that all of themolecules of a given protein have the samesequence.
• Proteins can be sequenced in two ways:
- real amino acid sequencing
- sequencing the corresponding DNA in the gene
Sequence Determination
Insulin consists of twopolypeptide chains, Aand B, held together bytwo disulfide bonds.The A chain has 21residues and the Bchain has 30 residues.
The sequence shown isthat of bovine insulin.
Determining the Sequence An Eight Step Strategy
• 1. If more than one polypeptide chain, separate.
• 2. Cleave (reduce) disulfide bridges
• 3. Determine composition of each chain
• 4. Determine N- and C-terminal residues
Determining the Sequence:
Determining the Sequence An Eight Step Strategy
• 5. Cleave each chain into smaller fragments and determine the sequence of each chain
• 6. Repeat step 5, using a different cleavage procedure to generate a different set of fragments.
Determining the Sequence:
Determining the Sequence An Eight Step Strategy
• 7. Reconstruct the sequence of the protein from the sequences of overlapping fragments
• 8. Determine the positions of the disulfide crosslinks
Determining the Sequence:
Step 1:Separation of chains
• Subunit interactions depend on weak forces
• Separation is achieved with:- extreme pH
- 8M urea
- 6M guanidine HCl
- high salt concentration (usually ammonium sulfate)
Step 1:
Step 2:
Cleavage of Disulfide bridges
• Performic acid oxidation
• Sulfhydryl reducing agents
- mercaptoethanol
- dithiothreitol or dithioerythritol
- to prevent recombination, follow with an alkylating agent like iodoacetate
Step 2:
Step 3:
Determine Amino Acid Composition
• described on pages 112,113 of G&G
• results often yield ideas for fragmentation ofthe polypeptide chains (Step 5, 6)
Step 3:
Step 4:
Identify N- and C-terminal residues
• N-terminal analysis:– Edman's reagent
– phenylisothiocyanate
– derivatives are phenylthiohydantions
– or PTH derivatives
Step 4:
Step 4:
Identify N- and C-terminal residues
• C-terminal analysis– Enzymatic analysis (carboxypeptidase)
– Carboxypeptidase A cleaves any residue exceptPro, Arg, and Lys
– Carboxypeptidase B (hog pancreas) only works onArg and Lys
Step 4:
Steps 5 and 6:
Fragmentation of the chains
• Enzymatic fragmentation– trypsin, chymotrypsin, clostripain,
staphylococcal protease
• Chemical fragmentation– cyanogen bromide
Steps 5 & 6:
Enzymatic Fragmentation
• Trypsin - cleavage on the C-side of Lys, Arg
• Chymotrypsin - C-side of Phe, Tyr, Trp
• Clostripain - like trypsin, but attacks Arg morethan Lys
• Staphylococcal protease– C-side of Glu, Asp in phosphate buffer
– specific for Glu in acetate or bicarbonate buffer
Enzymatic Fragmentation
Chemical Fragmentation
Cyanogen bromide
• CNBr acts only on methionine residues
• CNBr is useful because proteins usuallyhave only a few Met residues
• see Fig. 5.21 for mechanism
• be able to recognize the results!– a peptide with a C-terminal homoserine lactone
Chemical Fragmentation
Step 7:
Reconstructing the Sequence
• Use two or more fragmentation agents inseparate fragmentation experiments
• Sequence all the peptides produced (usuallyby Edman degradation)
• Compare and align overlapping peptidesequences to learn the sequence of theoriginal polypeptide chain
Step 7:
Reconstructing the Sequence
Compare cleavage by trypsin andstaphylococcal protease on a typical
peptide:
• Trypsin cleavage:
A-E-F-S-G-I-T-P-K L-V-G-K
• Staphylococcal protease:
F-S-G-I-T-P-K L-V-G-K-A-E
Reconstructing the Sequence
Reconstructing the Sequence
• The correct overlap of fragments:
L-V-G-K A-E-F-S-G-I-T-P-KL-V-G-K-A-E F-S-G-I-T-P-K
• Correct sequence:
L-V-G-K-A-E-F-S-G-I-T-P-K
Reconstructing the Sequence
Sequence analysis of catrocollastatin-C, a 23.6 kDprotein from the venom of Crotalus atrox
Nature of Protein Sequences
• Sequences and composition reflect thefunction of the protein
• Membrane proteins have more hydrophobicresidues, whereas fibrous proteins may haveatypical sequences
• Homologous proteins from differentorganisms have homologous sequences
• e.g., cytochrome c is highly conserved
Nature of Protein Seqences
Phylogeny of Cytochrome c
• The number of amino acid differencesbetween two cytochrome c sequences isproportional to the phylogenetic differencebetween the species from which they arederived
• This observation can be used to buildphylogenetic trees of proteins
• This is the basis for studies of molecularevolution
Phylogeny of Cytochrome C
Laboratory Synthesis of Peptides
• Strategies are complex because of the needto control side chain reactions
• Blocking groups must be added and laterremoved
• du Vigneaud’s synthesis of oxytocin in1953 was a milestone
• Bruce Merrifield’s solid phase method waseven more significant
Laboratory Synthesis of Peptides
Solid Phase Synthesis
• Carboxy terminus of a nascent peptide iscovalently anchored to an insoluble resin
• After each addition of a residue, the resinparticles are collected by filtration
• Automation and computer control nowpermit synthesis of peptides of 30 residuesor more
Laboratory Synthesis of Peptides