When sequencing proteins, one tries to generate overlapping peptides by using cleavages at specific sites. Which of the following statements about the cleavages caused by particular chemicals or enzymes is not true?
A. Cyanogen bromide cleaves at the carboxyl side of threonine
B. Trypsin cleaves at the carboxyl side of Lys and Arg
C. Chymotrypsin cleaves at the carboxyl side of aromatic and bulky amino acids
D. 2-Nitro-5-thiocyanobenzoate cleaves on the amino side of cysteine residues

Understanding Protein Cleavage in Protein Sequencing

Protein sequencing is a crucial method used in molecular biology to determine the amino acid sequence of a protein. The process often involves cleaving proteins at specific sites using chemicals or enzymes, generating smaller peptide fragments. These fragments are then sequenced to reconstruct the full protein sequence.

Certain enzymes and chemical agents cleave proteins at predictable amino acid sites, allowing researchers to generate overlapping peptide fragments for accurate sequencing. Understanding the specificity of these cleavage agents is essential for successful protein sequencing and analysis.

Correct Answer:

👉 The correct answer is A. Cyanogen bromide cleaves at the carboxyl side of threonine — This statement is false.

• Cyanogen bromide (CNBr) cleaves proteins at the carboxyl side of methionine (Met), not threonine (Thr).
• CNBr is widely used in protein sequencing due to its specificity for methionine residues.

Role of Protein Cleavage in Sequencing

To determine the sequence of a protein, it is often necessary to:
Break down the protein into smaller peptide fragments.
Use overlapping peptides to reconstruct the full sequence.
 Apply enzymes and chemicals with known specificity to target particular amino acid residues.

This strategy allows researchers to determine the order of amino acids and identify specific mutations or modifications in the protein.

Types of Protein Cleavage Agents

The following are common agents used for protein cleavage during sequencing:

1. Cyanogen Bromide (CNBr)

• Cleaves at the carboxyl side of methionine (Met) residues.
• Highly specific for methionine, making it useful for targeted protein fragmentation.
• CNBr forms a cyclic iminolactone intermediate, leading to cleavage at the methionine site.

Example:
A peptide sequence:
Ala-Met-Gly-Ser-Lys
After CNBr treatment:

• Cleaved into Ala and Met-Gly-Ser-Lys

2. Trypsin

• Cleaves at the carboxyl side of lysine (Lys) and arginine (Arg) residues.
• Does not cleave when proline (Pro) follows Lys or Arg.
• Trypsin is widely used due to its high specificity and efficiency in generating peptide fragments.

Example:
A peptide sequence:
Ala-Lys-Gly-Arg-Ser-Lys
After trypsin treatment:

• Cleaved into Ala-Lys, Gly-Arg, and Ser-Lys

3. Chymotrypsin

• Cleaves at the carboxyl side of aromatic (Phe, Trp, Tyr) and bulky amino acids (Leu).
• Chymotrypsin has a broad substrate specificity compared to trypsin.
• It functions optimally at an alkaline pH.

Example:
A peptide sequence:
Phe-Gly-Leu-Tyr-Arg
After chymotrypsin treatment:

• Cleaved into Phe, Gly-Leu, and Tyr-Arg

4. 2-Nitro-5-Thiocyanobenzoate (NTCB)

• Cleaves at the amino side of cysteine (Cys) residues.
• NTCB modifies cysteine residues to create a cleavage site.
• Useful in detecting disulfide bridges and cysteine-rich domains.

Example:
A peptide sequence:
Cys-Gly-Leu-Tyr-Arg
After NTCB treatment:

• Cleaved into Cys and Gly-Leu-Tyr-Arg

Explanation of Other Options

A. Cyanogen bromide cleaves at the carboxyl side of threonine

• Incorrect – Cyanogen bromide specifically cleaves at the carboxyl side of methionine, not threonine.
• Threonine is not targeted by CNBr due to its polar nature and hydroxyl group.

B. Trypsin cleaves at the carboxyl side of Lys and Arg

• Correct – Trypsin is highly specific for basic residues lysine and arginine.
• It does not cleave when proline follows these residues.

C. Chymotrypsin cleaves at the carboxyl side of aromatic and bulky amino acids

• Correct – Chymotrypsin targets aromatic residues like phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr).

D. 2-Nitro-5-Thiocyanobenzoate cleaves on the amino side of cysteine residues

• Correct – NTCB is used to specifically modify and cleave at cysteine residues.

Why Cyanogen Bromide is Important in Protein Sequencing

• Cyanogen bromide’s specificity for methionine makes it a valuable tool in protein sequencing.
• Since methionine is less frequently found in proteins, CNBr treatment creates large and manageable peptide fragments.

Applications of Protein Sequencing

1. Protein Structure Analysis:

   • Cleavage patterns provide insights into protein folding and stability.

2. Identification of Mutations:

   • Sequence changes can highlight disease-related mutations.

3. Post-Translational Modification:

   • Cleavage patterns can help identify phosphorylation, glycosylation, and other modifications.

4. Drug Target Identification:

   • Understanding protein structure helps in designing specific drugs.

Challenges in Protein Sequencing

• Contamination: Protein impurities can interfere with cleavage efficiency.
• Incomplete Digestion: Enzyme activity may vary based on pH and temperature.
• Complexity of Post-Translational Modifications: Modifications can alter cleavage patterns.

Conclusion

Protein sequencing relies on selective cleavage of proteins into manageable peptide fragments. Trypsin, chymotrypsin, cyanogen bromide, and NTCB are key agents used for this purpose. Cyanogen bromide specifically cleaves at the carboxyl side of methionine, not threonine, making option A incorrect. Understanding the specificity and mechanism of these cleavage agents enhances the accuracy and efficiency of protein sequencing, facilitating breakthroughs in biotechnology and medicine.