Question

5. (15 pts) You have been given a peptide for analysis. (1) Write out the six key steps to determine the sequence of a protein as discussed in the lecture. (2) What is the sequence of a peptide based on the following experimental results: (indicate your logic by placing a letter corresponding to the data set in parentheses above the amino acid in the sequence. This is required for full credit) a) Its amino acid composition is M+L+Y+C+S+2K. b) Treatment with Edman reagent releases phenylthiohydantoin(PTH)-cysteine c) Trypsin digestion yields a tetrapeptide, a dipeptide and free Leucine d) Digestion with chymotrypsin yields a pentapeptide and a dipeptide, both of which are basic. e) Cleavage with CNBr yields a neutral tripeptide and a basic tetrapeptide. . mer

Answer 1

Six key steps: 1. General protease digestion followed by analysis of the amino acids generated.

2. Digestion with trypsin (cuts after basic amino acids)

3. Digestion with chymotrypsin (cuts after aromatic amino acids)

4. Edman degradation (gives N terminal amino acid)

5. CNBr treatment (cuts after Methionine)

6. Carboxypeptidase treatment (gives C terminal amino acid)

The peptide sequence is C S M K Y K L

Given data set has 5 data, namely a, b, c, d, and e.

Edman gives cysteine (C) means it is the N terminal amino acid (b)

Trypsin cuts after K, at 4th and 6th position (c)

Chymotrypsin cuts after Y at 5th position (d)

CNBr cuts after Methionine (M) at 3rf position (e)