Introduction to Proteins: The Primary Level of Protein Structure

This section introduces the foundational concepts of protein structure, focusing on amino acids, peptide bonds, and the primary structure of polypeptides. It also covers essential laboratory techniques for protein purification and key terminology relevant to protein biochemistry.

Amino Acids: Structure, Classification, and Properties

• Definition: Amino acids are the building blocks of proteins, each containing an amino group, a carboxyl group, a hydrogen atom, and a unique side chain (R group) attached to a central (alpha) carbon.

• Chirality: Most amino acids (except glycine) are chiral, existing as L- or D-enantiomers. Proteins are composed exclusively of L-amino acids.

• Fischer Projection: A two-dimensional representation of molecules, where horizontal bonds project toward the viewer and vertical bonds project away.

• Zwitterion: At physiological pH, amino acids exist as zwitterions, carrying both a positive (amino group) and negative (carboxyl group) charge but are overall electrically neutral.

Classification of Amino Acids:

• Non-polar (hydrophobic): Side chains are mostly hydrocarbons (e.g., alanine, valine, leucine).

• Aromatic: Contain aromatic rings (e.g., phenylalanine, tyrosine, tryptophan).

• Polar (uncharged): Side chains can form hydrogen bonds (e.g., serine, threonine, asparagine).

• Charged: Side chains are positively (basic: lysine, arginine, histidine) or negatively (acidic: aspartate, glutamate) charged at physiological pH.

Codes for Amino Acids:

• Each amino acid has a three-letter and a one-letter code (e.g., Glycine: Gly, G).

Ionization and Isoelectric Point

• Ionizable Groups: Amino acids have groups that can gain or lose protons depending on pH (amino, carboxyl, and sometimes side chains).

• Charge Determination: The net charge of an amino acid or peptide at a given pH depends on the ionization state of its groups.

• Isoelectric Point (pI): The pH at which the molecule carries no net electrical charge.

Formula for Isoelectric Point (for amino acids without ionizable side chains):

Where and are the dissociation constants for the carboxyl and amino groups, respectively.

Peptide Bonds and Primary Structure

• Peptide Bond: A covalent bond formed between the carboxyl group of one amino acid and the amino group of another, releasing water (condensation reaction).

• Peptide Group: The six atoms (C=O, N-H, and the two alpha carbons) involved in the peptide bond are planar due to partial double-bond character, restricting rotation.

• Bond Distances: The C-N bond in a peptide is shorter than a typical single bond, reflecting its partial double-bond nature.

• Main Chain (Backbone): The repeating sequence of atoms in a polypeptide, excluding side chains.

• Amino Acid Residue: An amino acid unit within a polypeptide chain.

• Primary Structure: The unique linear sequence of amino acids in a polypeptide, defined by directionality (N-terminus to C-terminus) and individuality (specific sequence).

• N-terminus and C-terminus: The free amino group (N-terminus) and free carboxyl group (C-terminus) at the ends of a peptide or protein.

Example: The tripeptide Gly-Ala-Ser has glycine at the N-terminus and serine at the C-terminus.

Genetic Code and Protein Translation

• Gene Recognition: Identifying a gene involves finding an open reading frame (ORF) in a DNA sequence, which is a stretch of codons between a start and stop codon.

• Coding Strand: The DNA strand whose sequence matches the mRNA (except T for U).

• Non-coding Strand (Template): The DNA strand used as a template for mRNA synthesis.

• Translation: The process of converting the nucleotide sequence of mRNA into the amino acid sequence of a protein using the genetic code.

Example: Given a DNA sequence, the ORF is identified, and the corresponding amino acid sequence is determined using the genetic code table.

Protein Purification Techniques

Several chromatographic and electrophoretic methods are used to separate and analyze proteins based on their physical and chemical properties.

Size (Exclusion) Chromatography

• Principle: Separates proteins based on size by passing them through porous beads; larger proteins elute first because they are excluded from the pores.

• Order of Elution: Large proteins elute before small proteins.

Ion Exchange Chromatography

• Principle: Separates proteins based on charge by passing them over charged beads (anion or cation exchangers).

• Anion Exchange (e.g., DEAE): Beads are positively charged; negatively charged proteins bind and are eluted by increasing salt concentration.

• Cation Exchange (e.g., CM): Beads are negatively charged; positively charged proteins bind and are eluted by increasing salt concentration.

• Order of Elution: Proteins with the same charge as the beads elute first; those with opposite charge are retained and eluted later.

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

• Principle: Separates proteins by size under denaturing conditions; SDS binds proteins, giving them a uniform negative charge, so separation is based solely on size.

• Order of Migration: Smaller proteins migrate faster through the gel matrix.

Affinity Chromatography

• Principle: Utilizes specific binding interactions between a protein and a ligand attached to beads; only the protein of interest binds, while others wash through.

• Elution: The bound protein is eluted by adding a solution containing free ligand or by changing buffer conditions.

Key Terminology

Summary Table: Protein Purification Methods

Additional info: For exam preparation, students should be able to draw and recognize all 20 amino acids, understand their properties, and apply these concepts to protein structure and purification techniques.