Protein Structure, Modifications, and Analytical Techniques

Primary Structure and Amino Acid Properties

• Definition: The primary structure refers to the specific amino acid sequence of the protein.

• Stability: Stabilized by peptide bonds. Degradation requires harsh conditions: $6\,\text{N HCl}$ at $100 - 110^{\circ}\text{C}$ for $18 - 36\,\text{hrs}$.

• Hydrolysis Issues: Acid hydrolysis destroys $Trp$ and converts glutamine and asparagine into glutamic acid and aspartic acid.

• Symmetry: Amino acids typically exist in the L-amino acid absolute configuration.

• Acid-Base Properties: Proteins exist as zwitterions. Key $pK_a$ values include the carboxyl group ($\approx 2.1$) and the side chain of Histidine ($\approx 6.8$).

• Clinical Relevance: Cystinuria is a condition where incomplete reabsorption of cysteine in the kidney leads to high levels of cysteine and cystine in urine, forming stones.

Sequence Alignment and Evolution

• Analogy: Refers to sequences that are structurally similar but lack a demonstrated evolutionary relationship.

• Homology: Proteins with highly alignable sequences that belong to the same family, evolve from the same gene, and have similar functions.

• Paralog: Two homologous proteins present within the same species.

• Ortholog: Homologous proteins found in different species.

Protein Architecture: Secondary to Quaternary

• Peptide Bond: Features partial double bond character, is planar, and typically adopts a trans-configuration.

• Secondary Structure:

  • $\alpha$-helix: Right-handed coil with $3.6$ amino acids per turn, stabilized by H-bonds.

  • $\beta$-pleated sheets: Can be parallel or anti-parallel.

  • Turns/Loops: Type I and Type II $\beta$-turns are often rich in Glycine and Proline.

• Tertiary Structure:

  • Refers to the overall 3D conformation (location of each atom in space).

  • Stabilized by disulfide bonds (covalent) and non-covalent interactions (hydrophobic, H-bonds, and ionic).

• Quaternary Structure: Non-covalent assemblies of two or more monomer subunits.

• Domains: Distinct globular units within a protein that often possess different functions.

Denaturation and Folding

• Anfinsen Experiment: Demonstrated that the tertiary structure of globular proteins is determined by amino acid sequence using ribonuclease treated with urea and $\beta\text{-ME}$.

• Denaturing Agents:

  • Temperature: Disrupts hydrophobic interactions.

  • pH: Alters net charge, causing electrostatic repulsion and H-bond disruption.

  • Others: Organic solvents, detergents, urea, and heavy metal ions (Lead, Mercury).

Post-Translational Modifications

• Glycosylation: Provides protection against proteases and helps detect unfolded proteins. Malignant cells may show increased branching patterns.

• Ubiquitination: Involves E1, E2, and E3 ligases targeting proteins for degradation by the proteasome.

Analytical Techniques and Determination

• Sequence Determination: Edman Rxn uses Phenylisothiocyanate to identify N-terminal amino acids.

• Structural Methods:

  • Crystallography: Uses "salting out" to produce crystals; requires large samples.

  • NMR: No crystal needed, but limited to small proteins ($\approx 20\,\text{kDa}$).

  • Electron Cryomicroscopy: Used for 3D reconstruction; less effective for small fluctuations.

• Spectroscopic Properties: Fluorescence is used for cellular localization. Examples include DAPI and Green Fluorescent Protein (GFP) from the jellyfish Aequorea victoria.

• Electrophoresis and Chromatography:

  • SDS-PAGE: Separates proteins by mass/charge ratio (typically $5,000$ to $200,000\,\text{Da}$).

  • Western Blot: Uses antibodies for specific protein detection; can identify degradation or post-translational modifications.

  • Chromatography: Includes Ion Exchange, Gel Filtration, and Affinity methods.