Protein Structure and Function

Protein Structure

Protein Size and Composition

  • Proteins vary in size and shape.
  • They are large molecules composed of one or more polypeptide chains.
  • Proteins range from 35.5 to 220 kDa (kilodaltons), containing 50-2000 amino acid residues (mean molecular weight ≈ 100 Daltons).

Protein Functions

  • Enzymes: Catalyze chemical reactions.
  • Structural proteins: Provide mechanical strength.
  • Transport proteins: Carry molecules in the blood or into cells.
  • Motor proteins: Generate movement of cells and tissues.
  • Storage proteins: Bind small molecules.
  • Signal proteins: Carry signals between cells.
  • Receptor proteins: Detect and respond to signals.
  • Gene regulatory proteins: Control differentiation.

Levels of Protein Structure

  • Primary Structure: Amino acid sequence.
    • The primary amino acid sequence ultimately determines the final protein structure.
  • Secondary Structure: Repeating motifs.
    • α-helix
    • β-pleated sheet
  • Tertiary Structure: Overall folding of the polypeptide backbone and side chains.
  • Quaternary Structure: Interaction between multiple polypeptide chains.

Secondary Structure: α-Helix

  • Each oxygen of a carboxyl (-C=O) group from a peptide bond forms a hydrogen bond with the hydrogen atom attached to the amide nitrogen (-NH-) four amino acids down the peptide chain.
  • Structurally very stable.
  • Proline residues disrupt helix formation by introducing kinks.

Secondary Structure: β-Pleated Sheets

  • Multiple hydrogen bonds between peptide strands.
  • Can occur between different sections of the same long polypeptide.
  • Sections can be parallel or antiparallel, depending on their relative orientations.

Primary Structure and Secondary Structure

  • The sequence of amino acids and their side chains determines how the peptide chain folds.
  • Some sequences form α-helices, while others form β-pleated sheets.

Tertiary Structure

  • Overall folding of a polypeptide chain.
  • A protein molecule can have domains with α-helices and β-pleated sheets.
  • Other portions of the polypeptide chain may lack secondary structure.
  • Disulfide bonds (S-S) can form (depicted as yellow spheres).

Tertiary Structure Stability

  • Water-soluble proteins fold into compact structures with nonpolar cores.
  • Nonpolar side chains are in the hydrophobic core.
  • Polar side chains are on the outside of the molecule and can form hydrogen bonds with water.

Functional Domains

  • Domains are segments of the polypeptide chain that form stable, compact structures.

Depicting Protein Structure

  • Biochemists use different types of diagrams to depict protein structures:
    • Backbone
    • Ribbon
    • Wire frame
    • Space filling

Quaternary Structure

  • Some proteins contain two or more separate polypeptide chains.
  • Quaternary structure describes the form of the aggregate molecule.
  • Homodimers contain two identical subunits.
  • Heterodimers contain two different polypeptide chains.
  • Tetramers contain four polypeptide chains.

Bonds Between Polypeptide Subunits

  • Multiple weak, non-covalent bonds stabilize specific associations between large molecules.

Protein Assemblies

  • Binding of protein molecules can form larger assemblies, including filaments, tubes, and spheres.
  • Actin is a major intracellular protein that forms helical arrays.

Collagen

  • Collagen is a major component of the extracellular matrix.
  • It is a long, insoluble, fibrous protein that helps tissues withstand stretching.
  • The basic structural unit is a triple helix, with three polypeptide chains wrapped in a rope-like coil.

Collagen's Amino Acid Composition

  • Every third residue in each polypeptide is a glycine. These glycines are crucial for triple-helix formation.
  • Proline frequently follows glycine.
  • Proline is often modified by hydroxylation to hydroxyproline, providing additional hydrogen-bonding atoms to stabilize the helix.
  • The structure is called a Gly-Pro-X motif.

Vitamin C and Collagen

  • Vitamin C (ascorbic acid) is necessary for the hydroxylation of proline to form hydroxyproline.
  • Vitamin C deficiency results in collagen lacking sufficient hydroxyproline residues, so it cannot form as many hydrogen bonds.
  • This abnormal collagen is less stable and more readily broken down.
  • Clinical manifestations of Vitamin C deficiency (scurvy) include poor wound healing and fragility of small blood vessels (capillaries).

Collagen Fibrils and Fibers

  • Collagen fibrils are formed by cross-linking of many collagen triple helices.
  • Collagen fibers are larger macromolecular assemblies of collagen fibrils.
  • Linkages are formed by cross-linking of lysine residues, which are first modified to form hydroxylysine.

Elastin

  • Elastin is another component of the extracellular matrix.
  • Unlike collagen, it is able to stretch and relax.

Protein-Ligand Binding

  • Some proteins bind specifically to ligands.
  • Ligands can be small molecules, ions, or other macromolecules.
  • Many weak, non-covalent bonds combine to create the tight, high-affinity binding.
  • Hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions can contribute.
  • Protein folding brings together amino acids from different parts of the polypeptide chain to form a binding site.

Antibodies

  • Antibodies are soluble proteins that bind to foreign "invaders" during the body’s immune response.
  • Antibodies (e.g., IgG) contain four polypeptide chains: two heavy chains and two light chains, linked by disulfide bonds.
  • Each antibody has two distinct antigen-binding sites that recognize a particular molecular motif (antigen).
  • The portions that actually bind the antigen are called variable domains because they vary from one antibody to the next and provide for antibody specificity.

Prosthetic Groups

  • Many proteins contain non-amino acid components required for their specialized functions.
  • The amino acid portion is called an apoprotein.
  • Examples:
    • Heme component of myoglobin and hemoglobin is the oxygen-binding site.
    • Vitamin derivatives are key components of many enzymes.

Myoglobin and Hemoglobin

  • Myoglobin is the oxygen-binding protein within muscle cells.
  • It has a substantial amount of α-helical structure.
  • The heme group is a protoporphyrin molecule with an iron (Fe^{2+}) atom in the center.
  • Hemoglobin within red blood cells transports oxygen.
  • Each hemoglobin molecule has four polypeptide chains (2 α and 2 β).
  • Each polypeptide chain contains a heme group.
  • Binding of oxygen to the heme groups alters the quaternary structure of the protein.

Protein Denaturation

  • Denaturation is the process of disrupting the normal folded structure of a protein and separating its subunits.
  • Proteins are denatured by:
    • Heat
    • High salt concentrations
    • Urea and guanidinium chloride
    • Reducing agents (e.g., β-mercaptoethanol)
    • Organic solvents disrupt hydrophobic interactions

Denaturation of Ribonuclease

  • Ribonuclease is an enzyme that hydrolyzes RNA.
  • When treated with denaturing agents, it unfolds and loses its catalytic ability.
  • Denaturation can sometimes be reversible.

Protein Denaturation - Reversible vs. Irreversible

  • After denaturation, urea and mercaptoethanol can be removed by dialysis and the protein may regain its enzymatic activity.
  • Refolding does not proceed efficiently for many proteins, instead forming aggregates.
  • Insulin does not regain activity after denaturation because the molecule has two chains held together by –S-S- bonds which when broken, results in two irreversibly separated chains.

Disulfide Bonds in Hair

  • The protein keratin, which is the major component of hair, forms α-helical fibers.
  • Keratin fibers are crosslinked with many disulfide bonds.
  • Processes that curl or straighten hair break and re-form these cross-links.

Diseases Caused by Misfolded Proteins

  • Creutzfeldt-Jakob disease (Mad Cow Disease) is thought to be caused by misfolded proteins (prions).
  • Amyloid plaques associated with Alzheimer’s disease are misfolded proteins.

Protein Size & Separation

  • The mean molecular weight of an amino acid residue is approximately 100 Daltons.
  • Proteins with 50-2000 amino acid residues are between 5.5 and 220 kDa (kilodaltons).
  • Polyacrylamide gel electrophoresis separates proteins by size and eliminates effects of protein shape or net electrical charge.

Protein Charge & Isoelectric Point

  • The relative content of acidic and basic side chains determines the net electrical charge of a protein.
  • The isoelectric point (pI) of a protein is the pH at which it has a net charge of zero.
  • Isoelectric focusing separates proteins along a pH gradient until the pH equals their pI.
  • Serum albumin is an acidic protein with a pI of 4.8.