Protein Structure and Sequencing Methods

Protein Sequencing

• Protein sequencing is the process of determining the order of amino acids in a protein.
• Covalent structures of proteins and nucleic acids are important for protein sequencing.
• Proteins have various functions: enzymes (active sites for substrate binding), structural components (like creatinine, hemoglobin, myoglobin), and hormones (like peptide hormones and insulin).
• Proteins have four levels of structure.

Levels of Protein Structure

• Primary structure: The linear sequence of amino acids in a polypeptide chain.
• Secondary structure: Local folding of the polypeptide chain, stabilized by hydrogen bonds (e.g., alpha-helices, beta-pleated sheets).
• Tertiary structure: Three-dimensional folding of a protein due to side chain interactions (e.g., hydrogen bonds, ionic interactions, van der Waals forces, disulfide bonds).
• Quaternary structure: Protein consisting of more than one amino acid chain that interact. Insulin is an example.

Primary Structure

• Is the amino acid sequence
• Amino acids are connected by peptide bonds.
• Each amino acid unit is called a residue.

Determination of Primary Structure

• The primary structure is crucial for understanding a protein's 3D shape, function, and evolutionary relationships.
• Mutations in amino acid sequences can lead to diseases, providing insights for developing therapies.
• First complete amino acid sequence of a protein (insulin) was determined in 1953 using 100 grams. Now, it can be done using micrograms.

Sanger's Strategy

• For protein sequencing:
  • Preparation: Determine the number of subunits, cleave disulfide bridges, separate/purify polypeptide chains.
  • Fragmentation: Cleave chains into fragments less than 50 amino acids, then separate and purify the fragments.
  • Sequencing: Sequence the fragments using the Edman reagent.
  • Organization: Compare overlaps of fragments to determine the order and position of the disulfide bonds.

Protein Sequencing Example

• Purify a protein with 7 amino acids
• Expose to different proteolytic agents to obtain fragments.
• Fragments are used to determine the sequence based on overlaps.

Sequencing of Proteins

• Once the amino acid compositions are known, determine the specific sequence.
• Edman Degradation: A technique to determine the first amino acid.
• Exposure to phenyl isothiocyanate labels the first amino acid.

Cleaving Peptide Bonds

• Mildly acidic conditions cleave the first peptide bond, releasing the labeled amino acid.
• Repeated procedures determine the entire sequence.

Step 1: Preparation for Sequencing

• Identify the number of chains using SDS-PAGE.
• Identify N-terminal and C-terminal amino acids.
• Use methods like Sanger's method (DNFB), Dansyl chloride, PITC/Edman reagent, aminopeptidase (identifying N-terminus), and exopeptidases (identifying C-terminus).

Step 2: Fragmentation

• Use enzymes or chemicals (e.g., CNBr) to fragment polypeptide chains.
• Endopeptidases cleave peptide bonds within a protein's internal regions.

Specificity of Peptide Cleavage

• Enzymes like trypsin, chymotrypsin, elastase, and thermolysin have specificities for cleaving peptide bonds.
• Knowing their specificities gives insights into the sequence of the peptide.
• Reaction rates vary depending on the residue. This can impact the order of the amino acids.

Separation and Purification of Polypeptides

• Reduction of disulfide bonds is necessary to separate chains.
• Denaturation (heat) exposes SH groups to cleave the disulfide bonds.

Protection of Sulfhydryl Groups

• Alkylation: Preventing disulfide reformation by attaching a molecule (like iodoacetate) to the sulfur atom in the SH group.

Separation and Purification and Characterization of Polypeptide Chain

• Denaturation: Acidic-basic- low salt- high temperature- denaturant (urea-guanidin hypochloride- SDS)
• Separate chains by charge (IEC) or size (SEC-HPLC).
• Estimate the number of residues from the molecular weight (MW) using SEC-SDS-PAGE, and MS.

Step 2: Fragmentation of Polypeptide Chains

• Use enzymes (e.g., endopeptidases like trypsin) or chemicals (e.g., CNBr) to fragment the polypeptide chain.
• Endoproteases cleave internal peptide bonds.
• Some enzymes (e.g., trypsin, chymotrypsin) have specificities for cleaving peptide bonds after certain amino acid residues.

Ordering the Peptide Fragments

• Overlapping fragments from different cleavage points are compared.
• This provides information to establish the correct order of amino acids.

Identifying the Position of Disulfide Bonds

• This requires maintaining disulfide bonds during the cleavage process.
• Repeat fragmentation followed by separation (reverse phase HPLC) to identify disulfide bonds.

Determining Amino Acid Composition

• Employ hydrolysis (e.g., 6M HCl at high temperature) to break peptide bonds.
• Separate the amino acids via ion-exchange chromatography.
• Analyze the quantity of each amino acid using ninhydrin.

Example: Sequencing a Heptapeptide

• Provides illustration of the steps to determine the sequence of a small peptide using Sanger reagent, different enzyme treatments, and cleavage reactions (like by CNBr).