Protein Structure Notes

Protein Structure

Proteins are large molecules composed of one or more polypeptide chains, exhibiting a variety of sizes and shapes. A DNA molecule and a lipid bilayer are shown for size comparison.

Protein Functions

Proteins perform diverse functions including:

• 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 Structure in Proteins

• Primary: Amino acid sequence. The primary sequence dictates the final protein structure.

• Secondary: Repeating motifs like:

  • $\alpha$-helix

  • $\beta$-pleated sheet

• Tertiary: Overall folding of the polypeptide backbone and side chains.

• Quaternary: Interaction between multiple polypeptide chains.

Secondary Structure: $\alpha$-Helix

• Each oxygen of a carboxyl (-C=O) group forms a hydrogen bond with the hydrogen atom attached to the amide nitrogen (-NH-) four amino acids down the chain.

• Structurally very stable.

• Proline residues disrupt helix formation due to their structure.

$\beta$-Pleated Sheets

• Multiple hydrogen bonds between peptide strands.

• Can occur between different sections of the same long polypeptide.

• Sections can be parallel or anti-parallel based on their relative orientations.

Primary Structure and its Influence

The amino acid sequence with varying side chains dictates the folding of the peptide chain. Certain sequences favor $\alpha$-helices, while others form $\beta$-pleated sheets.

Tertiary Structure

• Overall folding of a polypeptide chain, forming domains with $\alpha$-helices and $\beta$-pleated sheets.

• Some portions may lack secondary structure.

• Disulfide bonds contribute to the tertiary structure.

Stability Provided by Tertiary Structure

Water-soluble proteins fold into compact structures with nonpolar cores. Polar side chains are on the outside, forming hydrogen bonds with water. Nonpolar side chains are in the hydrophobic core region.

Functional Domains

Domains are stable, compact structures within a polypeptide chain.

Depicting Protein Structure

Biochemists use different diagrams:

• Backbone

• Ribbon

• Wireframe

• Space-filling

Quaternary Structure

Some proteins have two or more separate polypeptide chains. Quaternary structure describes the arrangement of these aggregate molecules.

• Homodimers: Two identical subunits.

• Heterodimers: Two different polypeptide chains.

• Tetramers: Four polypeptide chains.

Bonds Between Polypeptide Subunits

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

Large Protein Assemblies

Proteins can form larger assemblies like filaments, tubes, and spheres. Actin, a major intracellular protein, forms helical filaments in the cytoskeleton.

Collagen Structure

Collagen is a major component of the extracellular matrix, providing strength to tissues.

• It's a long, insoluble, fibrous protein that helps tissues resist stretching.

• The basic unit is a triple helix with three polypeptide chains wrapped in a rope-like coil.

Unique Amino Acid Composition of Collagen

• Every third residue in each polypeptide is glycine, crucial for triple-helix formation.

• Proline frequently follows glycine and is often modified by hydroxylation to hydroxyproline, which provides additional hydrogen-bonding atoms to stabilize the helix.

• The structure is called a Gly-Pro-X motif.

Vitamin C Deficiency Effects on Collagen

Vitamin C (ascorbic acid) is needed for proline hydroxylation to form hydroxyproline.

A deficiency leads to collagen lacking sufficient hydroxyproline, reducing hydrogen bonds.

The abnormal collagen is less stable and breaks down more readily.
The 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 assemblies of collagen fibrils. Cross-linking of lysine residues, modified to hydroxylysine, forms these linkages.

Elastin vs. Collagen

Elastin is another component of the extracellular matrix.

Unlike rigid collagen, elastin can stretch and relax.

Protein Binding Specificity

Proteins can bind specifically to ligands.

Ligands can be small molecules, ions, or other macromolecules.

Many weak, non-covalent bonds combine to create tight, high-affinity binding.

Hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions all contribute.

Binding Site Formation

Protein folding brings together amino acids from different parts of the polypeptide chain.

Binding site formation brings together amino acid side chains that hydrogen bond with a small molecule like cyclic AMP.

Antibodies as Proteins

Antibodies are soluble proteins that bind to foreign "invaders" (bacteria, viruses, fungi, etc.) during the body’s immune response.

An IgG antibody contains four polypeptide chains: two heavy chains (blue) and two light chains (red), linked by disulfide bonds.

Each antibody has two antigen-binding sites that recognize a particular molecular motif (antigen) like a carbohydrate or protein.

Antibody Specificity

All antibodies of each class (like IgG) have a common structure and multiple domains in common.

Variable domains bind the antigen, varying from one antibody to the next, providing antibody specificity.

Prosthetic Groups

Many proteins contain non-amino acid components, called prosthetic groups, needed for their specialized functions.

The amino acid portion is called an apoprotein.

Examples:

• Heme in myoglobin and hemoglobin is the oxygen-binding site.

• Vitamin derivatives (like thiamin pyrophosphate and pyridoxal phosphate) are key components of many enzymes.

Myoglobin and Heme

Myoglobin is the oxygen-binding protein within muscle cells.

It is a polypeptide with significant $\alpha$-helical structure, folded into a globular, water-soluble shape.

The heme group is a protoporphyrin molecule with an iron ($Fe^{2+}$) atom in the center.

Hemoglobin

Hemoglobin within red blood cells transports oxygen.

Each hemoglobin molecule has four polypeptide chains (2 $\alpha$ and 2 $\beta$).

Each polypeptide chain contains a heme group.

Oxygen binding to heme groups alters the quaternary structure.

Protein Denaturation

Denaturation disrupts the normal folded structure of a protein and separates its subunits.

Proteins are denatured by:

• Heat

• High salt concentrations that disrupt electrostatic bonds.

• Urea and guanidinium chloride that disrupt non-covalent bonds.

• Reducing agents (like $\beta$-mercaptoethanol) that break disulfide bridges.

• Organic solvents that disrupt hydrophobic interactions.

Denaturation of Ribonuclease

Ribonuclease is an enzyme that hydrolyzes ribonucleic acid (RNA).

Denaturing agents unfold ribonuclease, causing it to lose catalytic ability.

Reversible Denaturation

After denaturation, urea and mercaptoethanol can be removed by dialysis.

Under suitable conditions, the protein regains enzymatic activity.

Sulfhydryl groups oxidize, forming disulfide bridges, and the molecule refolds into its original structure.

Irreversible Denaturation

Refolding isn't efficient for many proteins; molecules tangle and aggregate.

Insulin doesn't regain activity after denaturation.

Synthesized as one polypeptide, the mature molecule has two chains held together by -S-S- bonds.

Breaking the -S-S- bonds irreversibly separates the two chains.

Disulfide Bonds in Hair

The protein keratin, the major component of hair, forms $\alpha$-helical fibers.

Keratin fibers are crosslinked with many disulfide bonds.

Hair curling or straightening processes use a thiol-containing solution and heat to break a few cross-links.

Hair is curled or straightened, and an oxidizing agent is added to reform disulfide bonds, stabilizing the shape.

Diseases Caused by Misfolded Proteins

Jacobs-Kreuzfeld syndrome (Mad Cow Disease) is caused by misfolded proteins (prions) that propagate protein misfolding.

Amyloid plaques associated with Alzheimer’s disease are misfolded proteins.

Protein Sizes

The mean molecular weight of an amino acid residue is ~100 Daltons.

Proteins with 50 – 2000 amino acid residues are between 5.5 and 220 kd (kilodaltons).

Polyacrylamide gel electrophoresis separates proteins by size under denaturing conditions to eliminate effects of shape or charge.

Protein Electrical Charges

The relative content of acidic and basic side chains determines a protein's net electrical charge.

The isoelectric point (pI) of a protein is the pH at which it has a net charge of zero.

With isoelectric focusing, each protein moves along a pH gradient until the pH = its pI.

Serum albumin is an acidic protein with a pI of 4.8.