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.