Premed · Premed · Organic Chemistry 2
Lecture 19: Protein Structure
Organic Chemistry II
Learning Objectives
By the end of this lecture, students will be able to:
- Describe the four levels of protein structure (primary, secondary, tertiary, quaternary)
- Explain the forces that stabilize each level of protein structure
- Identify alpha-helices and beta-sheets and describe their hydrogen bonding patterns
- Describe the Ramachandran plot and its significance
- Explain the relationship between protein structure and function
- Describe protein denaturation and its causes
Lecture Content
I. Primary Structure
The primary structure of a protein is the linear sequence of amino acids in the polypeptide chain, read from the N-terminus to the C-terminus. This sequence is encoded by the gene (DNA sequence) for the protein, and the residues are connected by peptide bonds (amide bonds). The primary structure is the foundation upon which all higher levels of organization are built.
The seminal experiment demonstrating this principle was performed by Christian Anfinsen in 1961. He showed that ribonuclease A could be completely denatured and its disulfide bonds reduced, yet upon removal of the denaturant, the enzyme spontaneously refolded into its native, catalytically active conformation. This proved that the primary sequence alone contains all the information needed for correct three-dimensional folding, an insight recognized with the Nobel Prize in Chemistry in 1972.
Disulfide bonds (cystine bridges) are covalent S-S linkages formed by the oxidation of two cysteine residues. They can connect residues within the same polypeptide chain (intrachain) or between different chains (interchain), providing additional stabilization to the three-dimensional structure. Disulfide bonds are particularly important in extracellular proteins such as insulin and immunoglobulins, and they can be cleaved by reducing agents like beta-mercaptoethanol or DTT.
II. Secondary Structure
Secondary structure refers to the local folding patterns of the polypeptide backbone, stabilized by hydrogen bonds between backbone N-H groups and C=O groups.
The alpha-helix is a right-handed helical coil in which the C=O of residue i forms a hydrogen bond with the N-H of residue i+4. This i-to-i+4 pattern produces a helix with 3.6 residues per turn and a pitch (rise per turn) of 5.4 angstroms. The side chains project outward from the helix surface. Alpha-helices are abundant in both globular proteins (hemoglobin is approximately 75% alpha-helical) and fibrous proteins (keratin). Certain residues disrupt alpha-helices: proline introduces a kink because its cyclic side chain cannot accommodate the helical geometry and it lacks the N-H needed for hydrogen bonding, while glycine is too flexible to be consistently incorporated into a helix.
The beta-sheet (beta-pleated sheet) consists of extended polypeptide strands aligned side by side, connected by hydrogen bonds between adjacent strands. In a parallel beta-sheet, adjacent strands run in the same N-to-C direction; in an antiparallel beta-sheet, they run in opposite directions. Antiparallel sheets are slightly more stable because their hydrogen bonds are more linear. The side chains alternate above and below the plane of the sheet. Beta-sheets are prominent in silk fibroin and in many globular proteins.
Beta-turns (reverse turns) are tight four-residue turns that reverse the direction of the chain, typically connecting beta-strands. They are stabilized by a hydrogen bond between the C=O of residue i and the N-H of residue i+3. Proline and glycine are frequently found in beta-turns because proline facilitates the tight turn geometry and glycine fits in the constrained space. Loops and coils are irregular regions without repeating dihedral angles, but they are often functionally important, as they frequently form active sites and binding sites.
<image>Panel A: Alpha-helix shown as a ribbon diagram and as an atomic-level stick model. The hydrogen bonds between C=O of residue i and N-H of residue i+4 are depicted as dashed lines running nearly parallel to the helix axis. Key dimensions labeled: 3.6 residues per turn, 5.4 A pitch, 1.5 A rise per residue. R groups are shown projecting outward. Panel B: Antiparallel beta-sheet shown with two strands running in opposite directions, hydrogen bonds between strands depicted as dashed lines, R groups alternating above and below the sheet plane. Panel C: A beta-turn showing four residues with the i to i+3 hydrogen bond and typical proline at position i+1.</image>
III. The Ramachandran Plot
The Ramachandran plot maps the phi angle (rotation about the N-C-alpha bond) against the psi angle (rotation about the C-alpha-C bond) for each residue in a protein. Only certain combinations of phi and psi are sterically allowed, and these define the permitted regions of the plot. The alpha-helix occupies a characteristic region near phi = -57 degrees, psi = -47 degrees. Beta-sheets fall near phi = -120 degrees, psi = +120 degrees (antiparallel) or phi = -120 degrees, psi = +115 degrees (parallel). The left-handed alpha-helix region near phi = +60 degrees, psi = +60 degrees is sterically allowed but rarely populated, except by glycine.
Glycine, with no side chain beyond a hydrogen atom, enjoys the greatest conformational flexibility and populates regions of the Ramachandran plot that are forbidden to all other residues. Proline is the most restricted, with its cyclic side chain constraining phi to approximately -60 degrees. Ramachandran plots are routinely used to validate protein crystal structures; residues falling in disallowed regions suggest errors in the model.
IV. Tertiary Structure
Tertiary structure is the overall three-dimensional fold of a single polypeptide chain, determined by a combination of non-covalent forces and, in some cases, covalent disulfide bonds.
Hydrophobic interactions are the dominant driving force for protein folding. Nonpolar side chains cluster in the protein interior, away from the aqueous environment, driven by the hydrophobic effect (which increases the entropy of the surrounding water molecules). Hydrogen bonds form between side chain groups (for example, between the hydroxyl of serine and the carboxylate of aspartate) and between side chains and the backbone. Electrostatic interactions (salt bridges) pair oppositely charged side chains, such as the ammonium group of lysine with the carboxylate of aspartate. Van der Waals forces, though individually weak, are collectively important due to the close packing of atoms in the protein interior. Disulfide bonds are the only covalent interactions that contribute to tertiary structure.
Protein domains are independently folding units within a single polypeptide, often corresponding to functional modules such as immunoglobulin domains, kinase domains, or DNA-binding domains. Globular proteins are compact and spherical, with a hydrophobic core and a hydrophilic surface, making them water-soluble. Fibrous proteins, in contrast, are elongated, insoluble, and serve structural roles. Collagen, the most abundant protein in the human body, consists of a triple helix of three polypeptide chains with a repeating Gly-X-Y sequence (where X is often proline and Y is often hydroxyproline). Keratin forms alpha-helical coiled coils cross-linked by disulfide bonds and is found in hair, nails, and horns.
V. Quaternary Structure
Quaternary structure describes the arrangement of two or more polypeptide subunits in a multi-subunit protein complex. Not all proteins have quaternary structure; only those composed of multiple subunits exhibit this level of organization. The same non-covalent forces that stabilize tertiary structure -- hydrophobic interactions, hydrogen bonds, electrostatic interactions, and van der Waals forces -- operate at the subunit interfaces.
Hemoglobin is the classic example: an alpha2-beta2 tetramer with two alpha and two beta subunits, each containing a heme prosthetic group with an iron atom that binds oxygen. Hemoglobin exhibits cooperative binding, in which oxygen binding to one subunit increases the affinity of the remaining subunits, producing a sigmoidal binding curve. Other examples of quaternary structure include the triple helix of collagen, the multi-subunit complexes of DNA polymerase and RNA polymerase, and the highly symmetric protein shells of viral capsids.
<image>Four-panel illustration of the hierarchy of protein structure. Panel 1 (Primary): A linear amino acid sequence written as single-letter codes with peptide bonds connecting them. Panel 2 (Secondary): A ribbon diagram showing an alpha-helix and a beta-sheet with hydrogen bonds indicated. Panel 3 (Tertiary): A folded globular protein with hydrophobic core (shown in orange), salt bridges (shown as +/- pairs), disulfide bonds (S-S), hydrogen bonds, and van der Waals contacts labeled. Panel 4 (Quaternary): Hemoglobin structure showing four subunits (two alpha in blue, two beta in red) assembled together, each with a heme group (iron atom shown in the center of each heme).</image>
VI. Protein Denaturation
Denaturation is the loss of a protein's native three-dimensional structure -- its secondary, tertiary, and quaternary organization -- while the primary structure (the peptide bonds) remains intact. A variety of agents can cause denaturation. Heat disrupts hydrogen bonds and hydrophobic interactions. Extreme pH values alter the protonation states of side chains, disrupting salt bridges and hydrogen bonds. Organic solvents and chaotropic agents such as urea and guanidinium chloride disrupt hydrophobic interactions. Detergents like SDS bind to hydrophobic regions and unfold the protein. Reducing agents such as beta-mercaptoethanol and DTT break disulfide bonds. Heavy metal ions (Hg2+, Pb2+, Ag+) react with thiol groups and disrupt critical interactions.
The consequences of denaturation include loss of biological activity (an enzyme loses its catalytic function), altered solubility (denatured proteins often precipitate), and, in most cases, irreversibility. Cooking an egg is a familiar example of irreversible denaturation. However, as Anfinsen demonstrated, some proteins can refold reversibly under appropriate conditions.
Prions represent a pathological consequence of protein misfolding. The misfolded form of the prion protein (PrPSc) can template the conversion of normally folded prion protein (PrPC) into the misfolded state, causing transmissible spongiform encephalopathies such as mad cow disease and Creutzfeldt-Jakob disease. Prion diseases illustrate the critical importance of correct protein folding for biological function.
