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CHAPTER 5
Proteins: Their Biological Functions and Primary Structure
to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham
All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777 Copyright 1999 by Harcourt Brace & Company
Outline
5.1 Proteins - Linear Polymers of Amino Acids 5.2 Architecture 5.3 Many Biological Functions 5.4 May be Conjugated with Other Groups 5.7 Primary Structure Determination 5.8 Consider the Nature of Sequences
Biochemistry 2/e - Garrett & Grisham 5.1 Proteins are Linear Polymers of Amino Acids
Peptides
Short polymers of amino acids Each unit is called a residue 2 residues - dipeptide 3 residues - tripeptide 12-20 residues - oligopeptide many - polypeptide
Protein
One or more polypeptide chains
Biochemistry 2/e - Garrett & Grisham Configuration and conformation are not the same
Biochemistry 2/e - Garrett & Grisham Insulin consists of two polypeptide chains, A and B, held together by two disulfide bonds. The A chain has 21 residues and the B chain has 30 residues.
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)
Copyright 1999 by Harcourt Brace & Company
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 3:
Determine Amino Acid Composition described on pages 112,113 of G&G results often yield ideas for fragmentation of the polypeptide chains (Step 5, 6)
Step 4:
Identify N- and C-terminal residues N-terminal analysis:
Edman's reagent phenylisothiocyanate derivatives are phenylthiohydantions or PTH derivatives
Step 4:
Identify N- and C-terminal residues C-terminal analysis
Enzymatic analysis (carboxypeptidase) Carboxypeptidase A cleaves any residue except Pro, Arg, and Lys Carboxypeptidase B (hog pancreas) only works on Arg and Lys
Steps 5 and 6:
Fragmentation of the chains Enzymatic fragmentation
trypsin, chymotrypsin, clostripain, staphylococcal protease
Chemical fragmentation
cyanogen bromide
Enzymatic Fragmentation
Trypsin - cleavage on the C-side of Lys, Arg Chymotrypsin - C-side of Phe, Tyr, Trp Clostripain - like trypsin, but attacks Arg more than Lys Staphylococcal protease
C-side of Glu, Asp in phosphate buffer specific for Glu in acetate or bicarbonate buffer
Chemical Fragmentation
Cyanogen bromide CNBr acts only on methionine residues CNBr is useful because proteins usually have 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
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Step 7:
Reconstructing the Sequence Use two or more fragmentation agents in separate fragmentation experiments Sequence all the peptides produced (usually by Edman degradation) Compare and align overlapping peptide sequences to learn the sequence of the original polypeptide chain
Copyright 1999 by Harcourt Brace & Company
L-V-G-K-A-E-F-S-G-I-T-P-K
Sequence analysis of catrocollastatin-C, a 23.6 kD protein from the venom of Crotalus atrox
Phylogeny of Cytochrome c
The number of amino acid differences between two cytochrome c sequences is proportional to the phylogenetic difference between the species from which they are derived This observation can be used to build phylogenetic trees of proteins This is the basis for studies of molecular evolution
Copyright 1999 by Harcourt Brace & Company