Question

A sample of a peptide of unknown sequence was treated with trypsin; another sample of the same peptide was treated with chymotrypsin. The sequences (N-terminal to C-terminal) of the smaller peptides produced by trypsin digestion were as follows: $$Met-Val-Ser Thr-Lys$$ $$Val-Ile-Trp-Thr-Leu-Met-Ile$$ $$Leu-Phe-Asn-Glu-Ser-Arg$$ The sequences of the smaller peptides produced by chymotrypsin digestion were as follows: $$Asn-Glu-Ser-Arg-Val-Ile-Trp$$ $$Thr-Leu-Met-Ile$$ $$Met-Val-Ser-Thr-Lys-Leu-Phe$$ Deduce the sequence of the original peptide.

Short answer

Met-Val-Ser-Thr-Lys-Leu-Phe-Asn-Glu-Ser-Arg-Val-Ile-Trp-Thr-Leu-Met-Ile

Step-by-step solution

Understand the Role of Trypsin

Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine (K) or arginine (R). With this information in mind, note where trypsin digestion has occurred in each fragment.

Analyzing Trypsin Digest Fragments

For the sequences given by trypsin digestion: Met-Val-Ser Thr-Lys, Val-Ile-Trp-Thr-Leu-Met-Ile, Leu-Phe-Asn-Glu-Ser-Arg, the trypsin cleavage sites can be identified. Based on this, the original sequence before trypsin cleavage would have lysine or arginine after each of those fragments (where the cuts were made).

Understand the Role of Chymotrypsin

Chymotrypsin cleaves peptide chains mainly at the carboxyl side of phenylalanine (F), tryptophan (W), and tyrosine (Y), and sometimes leucine (L). Analyze the chymotrypsin digestion fragments based on these cleaving rules.

Analyzing Chymotrypsin Digest Fragments

For the sequences given by chymotrypsin digestion: Asn-Glu-Ser-Arg-Val-Ile-Trp, Thr-Leu-Met-Ile, Met-Val-Ser-Thr-Lys-Leu-Phe, understand where chymotrypsin cuts (after W, F, Y, and L) and where the original peptide sequence may consist of these cleavage sites.

Piece Together the Peptide Sequence

Combine the information from both digests to deduce the entire sequence. Start aligning the fragments to overlap at common endpoints, using the known cleavage sites.

Verify the Complete Sequence

Cross-check each part of the deduced sequence to ensure all fragments from both the trypsin and chymotrypsin digestions are included correctly. Make sure the sequences align properly at the cleavage points.

Key concepts

trypsin cleavage

Trypsin is an enzyme that plays a crucial role in protein digestion. It specifically cleaves peptide bonds on the carboxyl side of lysine (K) or arginine (R). This means that whenever trypsin encounters these amino acids in a protein sequence, it cuts the chain right after them.

In peptide sequencing, trypsin cleavage is used to break down a protein into smaller, more manageable fragments. By knowing where these cuts occur, we can deduce parts of the original sequence.

For example, if you have a fragment Met-Val-Ser-Thr-Lys after trypsin treatment, you know that Lys was a cleavage point. These predictable cut sites help in piecing together the full amino acid sequence of the original protein.

chymotrypsin cleavage

Chymotrypsin is another proteolytic enzyme, but it has different cleavage rules compared to trypsin. It cleaves on the carboxyl side of aromatic amino acids like phenylalanine (F), tryptophan (W), and tyrosine (Y), and sometimes at leucine (L).

When a peptide is subjected to chymotrypsin, the resulting fragments are cut around these specific amino acids. Knowing the pattern of chymotrypsin cuts can help deduce the positions of these amino acids in the original sequence.

In our example, consider the fragment Met-Val-Ser-Thr-Lys-Leu-Phe; chymotrypsin cuts after Phe (F), indicating that this amino acid was present in the intact protein at that specific point.

protein digestion

Protein digestion involves breaking down large protein molecules into smaller peptides and individual amino acids. Enzymes like trypsin and chymotrypsin are critical players in this process. They help in cleaving specific peptide bonds based on their substrate preferences.

By treating a protein with these enzymes, scientists generate distinct peptide fragments. Each enzyme has precise specificity, so the pattern of cleavage can provide valuable insights into the original sequence.

Understanding how these enzymes work allows us to map a protein’s primary structure thoroughly. This knowledge is essential for various applications, including protein identification and functional analysis.

amino acid sequence analysis

Amino acid sequence analysis is the process of determining the order of amino acids in a peptide or protein. This sequence is crucial as it defines the protein's structure and function.

In our example, by analyzing fragments produced by trypsin and chymotrypsin cleavage, we can piece together the original sequence. This process involves:

• Identifying consistent endpoints in fragments.
• Matching cleavage points from both enzymatic digests.
• Ensuring every part of the sequence fits together logically.

By overlapping fragments at specific cleavage points, we reconstruct the peptide's full amino acid sequence. This analysis is fundamental in studying protein functions, interactions, and in proteomics research.