6 - Protein Sequencing Notes

Objectives

  • Learn to determine a protein's primary sequence using Edman degradation, chemical, and enzymatic cleavage.
  • Understand the theory, general concepts, reagents, and enzymes to solve related problems.
  • Complete assigned homework problems.

Determining Protein Sequence

  • General Steps:
    • Hydrolyze the protein into constituent amino acids.
    • Use specific reagents to cleave the protein at specific sites.
    • Separate and identify individual amino acids.
    • Identify the N-terminal and C-terminal amino acids.
    • Determine sequences of smaller peptides.
    • Combine information from overlapping peptides to determine the complete sequence.

Determining Protein Composition

  • Process:

    • Hydrolyze the protein/peptide.
    • Use acid/heat to break it down.
    • Apply ion exchange chromatography. The amino acid concentration is proportional to the fluorescence.
    • React with the α-amino group.

  • Key Point:

    • This method determines the composition, not the sequence, of the protein.

Edman Degradation

  • Principle:

    • Determines one amino acid residue at a time from the N-terminus.

  • Steps:

    1. Treat the peptide with phenyl isothiocyanate (PITC), which reacts with the N-terminus to form a PTC-peptide.
    2. Treat with trifluoroacetic acid (TFA) to selectively cleave the N-terminal peptide bond.

  • Process Visualization:

    • Labeling: PITC labels the N-terminal amino acid.
    • Release: TFA cleaves the labeled amino acid.
    • The process repeats to sequence the next amino acid.

  • Chemical Reaction: Depicts the reaction of phenyl isothiocyanate with the N-terminal amino acid, followed by cleavage to release the PTH-amino acid and a shortened peptide.

  • Limitation:

    • Edman degradation can only reliably identify up to 50 amino acids in a peptide sequence.

Cleavage Methods for Longer Sequences

  • If the sequence is longer than 50 amino acids:
    • Fragmentation of proteins into smaller polypeptides is necessary using chemical or enzymatic methods, followed by Edman degradation.

A. Chemical Cleavage

  • Cyanogen bromide (CNBr): Cleaves peptide bonds after methionine residues.

B. Enzymatic Cleavages

  • Trypsin: Hydrolyzes peptide bonds after positively charged amino acids (lysine and arginine).
  • Chymotrypsin: Cleaves peptide bonds after phenylalanine, tyrosine, and tryptophan.

Specific Cleavage of Polypeptides

  • Chemical Cleavage

    • Cyanogen bromide: Carboxyl side of methionine residues.
    • O-Iodosobenzoate: Carboxyl side of tryptophan residues.
    • Hydroxylamine: Asparagine-glycine bonds.
    • 2-Nitro-5-thiocyanobenzoate: Amino side of cysteine residues.

  • Enzymatic Cleavage

    • Trypsin: Carboxyl side of lysine and arginine residues.
    • Clostripain: Carboxyl side of arginine residues.
    • Staphylococcal protease: Carboxyl side of aspartate and glutamate residues (glutamate only under certain conditions).
    • Thrombin: Carboxyl side of arginine.
    • Chymotrypsin: Carboxyl side of tyrosine, tryptophan, phenylalanine, leucine, and methionine.
    • Carboxypeptidase A: Amino side of carboxyl-terminal amino acid (not arginine, lysine, or proline).

Note: Focus on the underlined reagents/enzymes.

Cyanogen Bromide (CNBr) Mechanism

  • Cleaves peptide bonds after methionine residues.
  • CH3−S−C=NCH_3-S-C=N (Methylthiocyanate)

Trypsin Mechanism

  • Hydrolyzes peptide bonds after lysine and arginine.

  • Example:

    • Shows the cleavage sites of trypsin on a peptide sequence.

Chymotrypsin Mechanism

  • Cleaves after tyrosine, tryptophan, phenylalanine.

Example problem

  • Example shows how overlapping peptide sequences obtained by chymotrypsin and cyanogen bromide cleavage can be used to determine the overall sequence of a peptide.
  • Practice is key: Do the worksheet for practice.