Question

In the olden days (the \(1970 \mathrm{~s})\), the sequence of amino acids in a protein was determined by running a series of chemical reactions known as the Edman degradation. This technique worked only on short peptide chains, however, and large proteins had to be broken down into small parts in order to be sequenced. One way to chop large proteins into small chains was with enzymes known as proteases. The protease trypsin breaks the amide bond on the \(\mathrm{CO}_{2} \mathrm{H}\) side of arginine and lysine, and the protease chymotrypsin breaks the amide bond on the \(\mathrm{CO}_{2} \mathrm{H}\) side of tyrosine, phenylalanine, and tryptophan. An octapeptide treated with trypsin breaks down to the peptides Ala-Gly-Trp-Gly-Lys and Thr-Val-Lys, and the same octapeptide treated with chymotrypsin breaks down to the peptides Gly-Lys and Thr-Val-Lys-Ala-Gly-Trp. What is the sequence of the octapeptide?

Short answer

The sequence of the octapeptide is Ala-Gly-Trp-Gly-Lys-Thr-Val-Lys.

Step-by-step solution

Identify the breakdown products for each protease

Given the information, we have trypsin breaking the octapeptide into:\ Ala-Gly-Trp-Gly-Lys and Thr-Val-Lys. Chymotrypsin breaks the octapeptide into:\ Gly-Lys and Thr-Val-Lys-Ala-Gly-Trp.

Analyze the cleavage sites of both proteases

We know that: - trypsin breaks the amide bond on the CO2H side of arginine and lysine. - chymotrypsin breaks the amide bond on the CO2H side of tyrosine, phenylalanine, and tryptophan.

Determine overlapping peptide chains

From the breakdown products of trypsin, we can see that 'Gly-Lys' is common between Ala-Gly-Trp-Gly-Lys and Thr-Val-Lys. This means Ala-Gly-Trp-Gly-Lys and Thr-Val-Lys must overlap at 'Gly-Lys'. From the breakdown products of chymotrypsin, we can see that 'Thr-Val-Lys' is common between Gly-Lys and Thr-Val-Lys-Ala-Gly-Trp. This means Gly-Lys and Thr-Val-Lys-Ala-Gly-Trp overlap at 'Thr-Val-Lys'.

Combine the overlapping peptides to obtain the final sequence

We can now combine the overlapping peptides to obtain the original sequence of the octapeptide. From the trypsin breakdown, overlapping peptides give us:\ Ala-Gly-Trp-Gly-Lys-Thr-Val-Lys From the chymotrypsin breakdown, overlapping peptides give us:\ Thr-Val-Lys-Ala-Gly-Trp-Gly-Lys As we can see, both sequences give us the same octapeptide sequence:\ Ala-Gly-Trp-Gly-Lys-Thr-Val-Lys Thus, the sequence of the octapeptide is Ala-Gly-Trp-Gly-Lys-Thr-Val-Lys.

Key concepts

Edman Degradation

The Edman degradation is a well-known technique for sequencing amino acids in peptides. It was instrumental in making protein sequencing advancements during the 1970s. This process involves labeling and removing one amino acid at a time from the N-terminus of the peptide, allowing researchers to identify amino acids sequentially.

However, the method's limitation is that it works best with short peptide chains, typically up to 50 amino acids. For larger proteins, the structural complexity makes it less effective due to incomplete cycles or side reactions.

To overcome this hurdle, proteins would first be divided into smaller segments using proteolytic enzymes before employing Edman degradation. This segmenting allowed for a more manageable analysis of the protein's sequence by focusing on smaller, more defined peptide chains.

Proteases

Proteases are enzymes that play a crucial role in protein sequencing. They function by cleaving the peptide bonds within proteins, breaking them down into shorter fragments. This action is essential for various biological processes and laboratory applications.

In protein sequencing, specific proteases are used to target particular amino acid sequences. Different proteases cut at specific points, providing an intricate method to break down complex proteins into more manageable pieces. These smaller fragments can then be analyzed independently or further processed for sequencing methods like Edman degradation.

The combination of selective cleavage by proteases and subsequent analysis enables scientists to deduce the primary structure of proteins systematically.

Trypsin

Trypsin is a protease with a very specific function in proteomics. This enzyme cleaves the amide bonds on the carboxyl side of the amino acids arginine and lysine. This particular cleavage specificity is instrumental in generating predictable peptide maps of proteins being analyzed.

Often derived from the pancreas, trypsin is favored in laboratory settings because of its ability to operate under conditions that do not denature other proteins. Its precision makes it an invaluable tool for breaking down complex proteins into sizeable peptides that can be further examined using techniques such as Edman degradation.

Using trypsin in combination with other proteases enhances the accuracy of protein sequencing by providing different perspectives on the peptide structure.

Chymotrypsin

Chymotrypsin is another essential protease used in protein analysis. Unlike trypsin, chymotrypsin cleaves at different sites: it targets the carboxyl side of aromatic amino acids such as tyrosine, phenylalanine, and tryptophan. This specificity makes it particularly useful for studying proteins with a high content of aromatic amino acids.

Chymotrypsin, usually sourced from bovine pancreas, plays a complementary role when combined with other proteases, like trypsin. The varying cutting points offered by chymotrypsin allow for a comprehensive breakdown of the protein structure.

This enzyme's use, alongside trypsin, aids in covering more cleavage sites and ensures a thorough segmentation of proteins, which is essential for accurate mapping and sequencing. Chymotrypsin's unique selectivity is a powerful tool in the toolkit of protein biochemists.