BICH 410 Exam 2 Review 1-6-22

Exam 2 Review

Ch. 4: Amino Acids

• Structure of Amino Acids

  • Draw a generic amino acid, labeling:

    • α Carbon: central carbon atom.

    • Substituents: include amino group (−NH₂), carboxyl group (−COOH), hydrogen atom, and variable R group (side chain).

  • Structures of the 20 standard amino acids with one- and three-letter abbreviations:

    • E.g. Glycine (Gly, G), Alanine (Ala, A), etc.

  • Example tri-peptide: Cys–Gly–Asn

    • Peptide Bond: bond between amino acids.

    • N-terminus: amino end of the peptide.

    • C-terminus: carboxyl end of the peptide.

    • Net Charge at Neutral pH: calculate based on ionizable groups.

  • Classification of Amino Acids:

    • Polarity: Polar, Nonpolar, Charged (Acidic/Basic).

    • Structure: Branched, Aromatic, etc.

    • Functional Groups and Acid-Base Properties: classify according to ionization state, charge, etc.

Ch. 5: Primary Structure, Purification & Analysis

• Polypeptide Diversity:

  • Variable sequences arise from the combinations of the 20 amino acids.

  • Factors limiting polypeptide size: steric hindrance, solubility, and functionality.

  • Repetitive sequence in polypeptides: backbone sequence of atoms (N-Cα-C).

  • Peptide Bond Drawing: indicate donor and acceptor groups for hydrogen bonding.

• Protein Purification & Analysis:

  • Control environmental conditions (e.g. pH, temperature).

  • Quantification methods: assays (e.g. Bradford assay), absorbance spectroscopy (UV-Vis).

  • Salting Out: precipitation method based on solubility.

  • Antibody Usage: for purification (affinity chromatography) and concentration determination (ELISA).

  • Separation methods:

    • Ion Exchange Chromatography: based on charge.

    • Gel Filtration Chromatography: based on size.

    • Affinity Chromatography: based on specific interactions.

  • Electrophoresis:

    • SDS-PAGE: separates denatured proteins by size.

    • 2D Gel Electrophoresis: separates based on isoelectric point and size.

  • Ultracentrifugation: information on molecular weight and sedimentation coefficients.

• Protein Sequencing:

  • Steps in sequencing: cleaving, separation, analysis.

  • Importance of N-terminal residue identification: aids in sequencing methods.

  • Mass Spectrometry Advantages: faster than Edman degradation.

  • Long polypeptides require fragmentation for effective sequencing.

  • Protein Sequence Databases: comparative analysis, functional predictions.

Ch. 6: Protein Structure

• Hierarchy of Protein Structure:

  • Primary: sequence of amino acids.

  • Secondary: local folding (e.g. α-helix, β-sheets).

  • Tertiary: complete 3D shape of a single polypeptide.

  • Quaternary: assembly of multiple polypeptides.

  • Not all proteins have all four levels; some function at the tertiary level only.

• Conformational Limitations:

  • Peptide bond rigidity influences folding patterns.

  • Features of α-helices and β-sheets: stabilization through hydrogen bonding.

  • Fibrous Proteins: unique properties from secondary structure.

    • Example structures:

      • α Keratin: coiled coil, heptad repeat.

      • β Keratin: stacked β-sheet, alternate repeating units.

      • Collagen: triple helix, specific repeats for strength.

• Tertiary Structure:

  • Determines functionality based on amino acid sequence.

  • Surface vs Interior Side Chains: hydrophilic on the surface, hydrophobic inside.

  • Common protein motifs: combinations of secondary structure elements.

• Quaternary Structure:

  • Benefits of multiple subunits: regulation, stability.

  • No mirror symmetry due to chirality of amino acids.

• Protein Stability:

  • Stabilizing forces: hydrogen bonds, hydrophobic interactions, ionic interactions.

  • Denaturation: loss of structure due to external agents (temperature, pH).

  • Concept of Tm (melting temperature).

  • Discussion on whether it is possible to "unscramble an egg" when proteins denature.

• Protein Folding:

  • Energy and entropy changes during folding favor lower energy states.

  • Renaturation differs in vitro vs in vivo (environmental conditions).

  • Role of ATP and chaperones (Hsp70, GroEL/ES) in proper protein folding.

  • Amyloid diseases related to aging due to misfolded proteins.

Ch. 7: Protein Function

• Hemoglobin vs Myoglobin:

  • Myoglobin (Mb): O2 storage, no regulation.

  • Hemoglobin (Hb): O2 transport, allosterically regulated by

    • CO2,

    • H+, and

    • BPG (2,3-bisphosphoglycerate).

  • Fetal Hemoglobin: differs in subunit composition, binds O2 tighter.

  • Sickle-Cell Anemia: mutation in β-subunit leading to polymerization (E6V), resulting in sickling of red blood cells and anemia.

  • Selective pressure for Hb S due to malaria resistance.