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BCH210H1 (352)
Lecture 3

Lecture 3 protein sequencing

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Stavroula Andreopoulos

Protein Sequencing - amino acid sequence is read from N-terminal to C-terminal on a polypeptide chain - chemical strategy for determining amino acid sequence of a protein has six steps” 1. If the protein contains more than one polypeptide chain, he chains are separated and purified. 2. Intrachain disulfide cross-bridges between cysteine residues in the polypeptide chain are cleaved. If the disulfides are interchain linkages, then step 2 precedes the first step. 3. The N-terminal and C-terminal residues are identified. 4. Each polypeptide chain is cleaved into smaller fragments, and the amino acid composition and sequence of each fragment are determined. 5. Step 4 is repeated, using a different cleavage procedure to generate a different and therefore overlapping set of peptide fragments. 6. The overall amino acid sequence of the protein is reconstructed from the sequences in overlapping fragments. Step 1 – Separation of Polypeptide Chains - if protein is heteromultimer, it must be dissociated into its polypeptide chains - polypeptide chains must be separated from one another and sequenced individually by size/charge - some heteromultimers are linked by interchain disulfide bridges – crosslinks must be cleaved before dissociation and isolation of chains Step 2 – Cleavage of Disulfide Bridges - disulfide bridges must be cleaved in a way so that the original or new disulfide bridges do not form - oxidation of a disulfide by performic acid forms two equivalents of cysteic acid – electrostatic repulsion prevents disulfide recombination - sulfhydryl compounds also reduce disulfide bridges to two cysteine side chains – may create other disulfide bonds – alkylating agents prevent recombination by modifying the SH groups and blocking disulfide bridge formation Step 3A – N-terminal Analysis - the amino acid at the N-terminal can be identified by Edman degradation – identifies the sequence starting at the N-terminal - phenylisothiocyanate (Edman reagent) combines with
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