5.4 How to determine the primary structure of a protein

Determining Primary Structure of a Protein

• Determining the primary structure of a protein involves several key steps to identify the amino acids present and their sequence.

Amino Acid Composition Analysis

• Before sequencing, determine which amino acids are present and their proportions.
• Hydrolyze the protein into individual amino acids using heat in 6M hydrochloric acid (HCl) at 100–110°C for 12–36 hours, which breaks all peptide bonds.
• Separate and identify the resulting amino acid mixture using ion-exchange chromatography or high-performance liquid chromatography (HPLC).
• An amino acid analyzer provides qualitative data (which amino acids are present) and quantitative data (how much of each amino acid is present).
• This step guides sequencing by identifying rare or modified amino acids like hydroxyproline.

Identification of N-terminal and C-terminal Amino Acids

• Determines where the protein sequence starts and ends.
• Verifies whether the protein consists of one or multiple polypeptide chains.

N-terminal Identification

• Edman degradation uses phenyl isothiocyanate (PITC), which binds to the first amino acid and releases it as a stable phenylthiohydantoin (PTH) derivative detectable chromatographically.

C-terminal Identification

• Carboxypeptidases are enzymes that selectively cleave one amino acid at a time from the C-terminus.
• This step is less common now, modern sequencing techniques can directly determine the full sequence without needing separate N- and C-terminal analysis.

Cleavage of Protein into Peptides

• Long polypeptide chains (100+ residues) are broken into smaller fragments (20–50 amino acids each) for easier sequencing.

Enzymatic Cleavage

• Specific proteolytic enzymes (proteases) cleave peptide bonds at defined sites.

Trypsin Specifics

• Trypsin cleaves at the carboxyl side of basic amino acids like Lysine (Lys, K) and Arginine (Arg, R).
• For example, Ala—Arg—Gly—Lys—Phe is cleaved into Ala—Arg, Gly—Lys, and Phe.
• Results in peptides with positively charged residues (Lys, Arg) at the C-terminal end.
• It is a proteolytic enzyme that cleaves only peptide bonds where arginine or lysine contributes the carbonyl function.

Chymotrypsin Specifics

• Cleaves at the carboxyl side of aromatic amino acids like Tyrosine (Tyr, Y), Tryptophan (Trp, W), and Phenylalanine (Phe, F).
• For example, Ser—Tyr—Gly—Trp—Leu—Phe is cleaved into Ser—Tyr, Gly—Trp, and Leu—Phe.
• Chymotrypsin hydrolyzes proteins at aromatic amino acids.

Reason for Multiple Cleavage Methods

• Different methods generate overlapping peptide fragments, allowing reconstruction of the full sequence.
• Trypsin cleaves at Arg and Lys, producing certain peptides, while chymotrypsin cleaves at Tyr, Trp, and Phe, producing different peptides.
• Overlapping fragments from both methods help piece together the original protein sequence.
• Once peptides are obtained, they are separated and purified using chromatography techniques like HPLC.

Chemical Cleavage

• Some chemical reagents can break peptide bonds at specific amino acid residues.

Cyanogen Bromide (CNBr)

• Cleaves at methionine (Met, M) residues.
• Produces peptides where C-terminal ends are homoserine lactone.
• For example: Val-Met-Ala-Gly is cleaved into Val-Met and Ala-Gly.

Determining the Amino Acid Sequence

• The most common method for sequencing peptides (10–40 residues long) is Edman Degradation.
• Phenyl isothiocyanate (PITC) is used to modify the N-terminal amino acid, which is then selectively cleaved and identified.

Steps of Edman Degradation

• PITC binds to the N-terminal amino acid.
• The modified residue is cleaved off without breaking the rest of the peptide.
• The released amino acid is detected using HPLC.
• The process is repeated for the next amino acid to determine the sequence step-by-step.

Limitations of Edman Degradation

• It can only sequence up to ~40 amino acids before errors accumulate.
• It is ineffective if the N-terminus is blocked by chemical modifications.

Mechanism of Peptide Sequencing by Edman Degradation

• Phenylisothiocyanate interacts with the N-terminus of a peptide under mildly alkaline conditions, resulting in a phenylthiocarbamoyl substitution.
• Treatment with trifluoroacetic acid (TFA) causes cyclization, leading to the release of the N-terminal amino acid residue as a thiazolinone derivative, leaving other peptide bonds intact.
• The N-terminal amino acid is then obtained as a phenylthiohydantoin (PTH) derivative through organic extraction and processing with aqueous acid.
• This sequence of steps is reiterated to reveal the N-terminus at subsequent phases until the full composition of the peptide is ascertained.

DNA Sequencing as an Alternative Method

• Instead of sequencing the protein directly, the gene encoding the protein can be sequenced using modern DNA sequencing techniques.

Advantages of DNA Sequencing

• Faster and more efficient than protein sequencing.
• Can predict the entire protein sequence using the genetic code.

Limitations of DNA Sequencing

• Post-translational modifications (PTMs) are not detected like disulfide bonds, phosphorylation, and glycosylation.
• Alternative splicing may create different protein variants not reflected in the gene sequence.