Question

Three peptides were obtained from a trypsin digestion of two different polypeptides. In each case, indicate the possible sequences from the given data and tell what further experiment should be carried out in order to determine the primary structure of the polypeptide.
a. polypeptide I:
1. Val-Gly-Asp-Lys
2. Leu-Glu-Pro-Ala-Arg
3. Ala-Leu-Gly-Asp

Accepted Answer

Step 1: Understand the problem. Trypsin is a proteolytic enzyme that cleaves peptide bonds specifically at the carboxyl side of lysine (Lys) and arginine (Arg) residues, unless they are followed by proline (Pro). This means the given peptides are fragments resulting from trypsin digestion of polypeptide I. Step 2: Analyze the given peptide fragments. The three fragments provided are: (1) Val-Gly-Asp-Lys, (2) Leu-Glu-Pro-Ala-Arg, and (3) Ala-Leu-Gly-Asp. Note that fragments (1) and (2) end with Lys and Arg, respectively, which are cleavage sites for trypsin. Step 3: Determine the possible sequence of the polypeptide. Since trypsin cleaves at Lys and Arg, the original polypeptide sequence must have had these residues at the cleavage points. To reconstruct the sequence, look for overlaps or logical connections between the fragments. For example, fragment (3) does not end with Lys or Arg, so it must be at the N-terminal or connected to another fragment. Step 4: Propose further experiments. To confirm the primary structure of the polypeptide, perform Edman degradation on each peptide fragment to determine the sequence of amino acids. Additionally, mass spectrometry can be used to determine the molecular weight of the fragments and confirm their sequences. Step 5: Consider overlapping peptide analysis. To further refine the sequence, perform partial digestion with another protease (e.g., chymotrypsin) to generate overlapping fragments. This will help establish the order of the fragments in the original polypeptide.

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Key Concepts

Peptide Sequencing

Trypsin Digestion

Primary Structure Determination