A scientist was trying to deduce the amino acid sequence of

A scientist was trying to deduce the amino acid sequence of a short polypeptide. He tried to use Edman degradation but the machine broke after the first round. The residue identified with the first round was Ala. He then decided to use various proteases and chemical reagents to cleave the short peptide and identify the amino acids using his HPLC machine. Using the data below, the scientist was able to deduce the amino acid sequence. What is the primary sequence of the peptide (please denote the N- and C- termini)? Explain each piece of evidence given and the reasoning that led to your answer. (Use your textbook (Page 73-79) or other sources (i.e. lecture notes) to help you identify cleavage sites) Acid hydrolysis: Alax2, Arg, Lys x2, Met, Phe, Ser x2 Carboxypeptidase A: (Ala) Trypsin: (Ala, Arg) (Lys, Phe, Ser) (Lys) (Ala, Met, Ser) CNBr: (Ala, Arg, Lys x2, Met, Phe, Ser) (Ala, Ser) Chymotrypsin: (Phe, Arg, Ser, Ala) (Met, Ser, Lys x2, Ala).

Solution

A. This protein contains 9 amino acids. Acid hydrolysis breaks down the CO-NH peptide bond present between two amino acids. This will result in individual amino acids present in the protein.

B. At the carboxy terminal end, \'Ala\' is present. This is so because carboxypeptidase cleaves the C-terminal of a protein.

C. Trypsin cleavage sites are lysine and arginine. Resuts are showing four strands. So, number of cuts are 3. The results also prove that Ala is present on the N-terminal as well.

Ala-Arg-(1st cut)-Ser-Phe-Lys-(2nd cut)-Lys-(3rd cut)-Met-Ser-Ala

D. CNBr always cleaves at Met residues. The number of strands is 2; this means that one Met is there in the protein.

E. Please note that chymotrypsin selectively cleaves peptide bonds formed by aromatic residues (tyrosine, phenylalanine, and tryptophan). Two residues mean one aromatic amino acid is there; Phe is there. Sequence becomes:

Ala-Arg--Ser-Phe-Lys-Lys-Met-Ser-Ala