Premed · Premed · Biochemistry
Lecture 4: Protein Structure: Primary Through Quaternary
Biochemistry
Learning Objectives
By the end of this lecture, students will be able to:
- Describe the four levels of protein structure and the forces that stabilize each level
- Explain how the Ramachandran plot describes allowed backbone conformations
- Identify and describe the major types of secondary structure (alpha-helix, beta-sheet, turns)
- Distinguish between structural motifs, domains, and subunits
- Provide examples of fibrous and globular proteins and their structural features
- Explain how quaternary structure contributes to protein function
Lecture Content
I. Overview of the Four Levels of Protein Structure
Protein structure is organized into four hierarchical levels. Primary structure is the linear amino acid sequence. Secondary structure refers to local folding patterns such as alpha-helices and beta-sheets. Tertiary structure describes the overall three-dimensional shape of a single polypeptide chain. Quaternary structure is the arrangement of multiple polypeptide subunits. Each level builds on the previous, and higher-order structure is ultimately determined by the primary sequence.
II. Primary Structure
The primary structure is the amino acid sequence of a polypeptide chain, read from the N-terminus to the C-terminus. It is determined by the gene encoding the protein and dictates all higher levels of structure. Even a single amino acid change can have dramatic consequences, as illustrated by sickle cell disease, in which a glutamate at position 6 of beta-globin is replaced by valine. This substitution swaps a charged, hydrophilic residue for a nonpolar, hydrophobic one, causing hemoglobin to polymerize under low-oxygen conditions. The conservation of sequences across homologous proteins in different species reflects the functional importance of particular residues.
III. The Ramachandran Plot
The backbone conformation of a polypeptide is defined by the dihedral angles phi (rotation around the N-Calpha bond), psi (rotation around the Calpha-C bond), and omega (rotation around the C-N peptide bond, usually fixed at approximately 180 degrees in the trans configuration). The Ramachandran plot maps the allowed combinations of phi and psi angles, revealing that many combinations are sterically forbidden because atoms would clash. The allowed regions correspond to regular secondary structures: the alpha-helix occupies the region near phi approximately -57 degrees and psi approximately -47 degrees, while the beta-sheet falls near phi approximately -120 degrees and psi approximately +130 degrees. Glycine has the most conformational flexibility because its small R group creates fewer steric clashes, while proline is the most restricted because its cyclic side chain constrains phi to approximately -60 degrees.
IV. Secondary Structure
Alpha-Helix
The alpha-helix is a right-handed helical structure with 3.6 residues per turn and a rise of 5.4 Angstroms per turn (1.5 Angstroms per residue). It is stabilized by hydrogen bonds between the C=O group of residue i and the N-H group of residue i+4, with these hydrogen bonds running parallel to the helix axis. The R groups project outward from the helix. Amino acids with favorable phi/psi angles, such as alanine, leucine, methionine, glutamate, and lysine, are alpha-helix formers, while proline (which introduces a kink and cannot donate an H-bond) and glycine (which is too flexible) tend to break helices. Alpha-helices are found extensively in fibrous proteins like keratin, in coiled-coil structures, and in membrane-spanning regions. Amphipathic helices, with one hydrophobic face and one hydrophilic face, are commonly found at membrane surfaces.
Beta-Sheet
Beta-sheets consist of extended polypeptide chains (beta-strands) aligned side by side and stabilized by hydrogen bonds between C=O and N-H groups of adjacent strands, with these hydrogen bonds running perpendicular to the strand direction. In parallel beta-sheets, the strands run in the same direction and the hydrogen bonds are slightly angled. In antiparallel beta-sheets, the strands run in opposite directions and the hydrogen bonds are straight, making the structure more stable. The R groups alternate above and below the sheet plane. Beta-sheets are found in many structural proteins such as silk fibroin and are common in enzyme cores.
Turns and Loops
Beta-turns (reverse turns) connect adjacent strands of antiparallel beta-sheets and usually involve 4 residues stabilized by a hydrogen bond between the C=O of residue i and the N-H of residue i+3. Glycine and proline are commonly found in turns. Loops are less regular regions of variable length, often located on the protein surface, where they frequently participate in ligand binding and protein-protein interactions. Loops tend to be more variable in sequence between homologous proteins.
<image>A three-panel figure illustrating secondary structures. Panel A: An alpha-helix shown as both a ribbon diagram and a ball-and-stick model, with hydrogen bonds drawn as dashed lines between i and i+4 residues, dimensions labeled (3.6 residues/turn, 5.4 A pitch). Panel B: Parallel and antiparallel beta-sheets shown side by side, with arrows indicating strand direction and hydrogen bonds drawn between strands. Panel C: A beta-turn connecting two antiparallel strands, showing the i to i+3 hydrogen bond and the common positions of Gly and Pro residues.</image>
V. Supersecondary Structures (Motifs)
Certain combinations of secondary structure elements recur throughout nature. The beta-alpha-beta motif consists of two parallel beta-strands connected by an alpha-helix. The beta-hairpin features two antiparallel beta-strands connected by a tight turn. The helix-turn-helix motif is found in DNA-binding proteins. The coiled-coil consists of two alpha-helices wound around each other, as seen in the leucine zipper. The Greek key motif involves four antiparallel beta-strands folded in a specific pattern. The beta-barrel is a beta-sheet rolled into a barrel shape, found in porins and other membrane proteins.
VI. Tertiary Structure
Tertiary structure is the complete three-dimensional arrangement of all atoms in a single polypeptide chain, encompassing all secondary structure elements plus the connecting loops. It is stabilized by multiple forces. Hydrophobic interactions, in which nonpolar side chains pack together in the protein interior, are the major driving force. Hydrogen bonds form between side chains and between side chains and the backbone. Ionic interactions (salt bridges) form between oppositely charged side chains such as lysine and aspartate or arginine and glutamate. Van der Waals forces arise from the close packing of atoms in the interior. Disulfide bonds, covalent linkages between cysteine residues, are common in extracellular proteins where they stabilize structure in the oxidizing environment.
Domains are independently folding units within a protein, each typically having a distinct function such as binding or catalysis. Domains are connected by flexible linker regions and can be identified experimentally through limited proteolysis.
VII. Fibrous vs. Globular Proteins
Fibrous Proteins
Fibrous proteins have elongated, rope-like structures that provide structural support and typically contain repeating sequences and regular secondary structure. Alpha-keratin is a coiled-coil of alpha-helices found in hair, nails, and skin, cross-linked by disulfide bonds (more disulfide bonds make the structure harder). Collagen is the most abundant protein in the body, forming a triple helix of three polyproline II-like chains with a characteristic Gly-X-Y repeat (where X is often proline and Y is often hydroxyproline). Glycine must occur at every third position because it is the only amino acid small enough to face the interior of the triple helix. The structure is stabilized by hydrogen bonds between chains. Silk fibroin consists of stacked antiparallel beta-sheets with the repeating sequence Gly-Ser-Gly-Ala-Gly-Ala.
Globular Proteins
Globular proteins are compact and roughly spherical with a hydrophobic core and hydrophilic surface. They serve diverse functions as enzymes, transport proteins, antibodies, and receptors. Examples include hemoglobin, myoglobin, and immunoglobulins.
<image>A comparison figure of fibrous and globular protein structures. Panel A: Alpha-keratin shown as a coiled-coil of two alpha-helices with disulfide cross-links indicated. Panel B: Collagen triple helix showing the three chains wound around each other, with a close-up of the Gly-X-Y repeat and the position of glycine in the center. Panel C: A globular protein (myoglobin) shown as a ribbon diagram with its hydrophobic core shaded and the heme prosthetic group visible. A cutaway view shows the nonpolar residues packed in the interior and polar residues on the surface.</image>
VIII. Quaternary Structure
Quaternary structure describes the spatial arrangement of two or more polypeptide subunits (protomers) in a multi-subunit protein. Not all proteins have quaternary structure -- only those composed of multiple subunits. The same non-covalent forces that stabilize tertiary structure also stabilize subunit interfaces, including hydrophobic interactions, hydrogen bonds, ionic interactions, and van der Waals forces. Some proteins also have disulfide bonds between subunits, as seen in immunoglobulins.
Subunit composition is described using Greek letters: a homodimer consists of two identical subunits (alpha2), while a heterotetramer such as hemoglobin has the composition alpha2-beta2. Multi-subunit proteins offer several advantages: economy of genetic material (one gene can produce a symmetric complex), cooperativity in ligand binding (as in hemoglobin), allosteric regulation, and the ability to bring catalytic sites into proximity in multienzyme complexes.
Notable examples include hemoglobin (alpha2-beta2), which exhibits cooperative oxygen binding; lactate dehydrogenase, a tetramer of M and H subunits that forms tissue-specific isozymes; and DNA polymerase III, a complex holoenzyme with multiple subunits.
<image>A diagram showing quaternary structure using hemoglobin as an example. Panel A: The individual alpha and beta subunits shown separately as ribbon diagrams, each with a heme group. Panel B: The assembled alpha2-beta2 tetramer showing the subunit interfaces and the central cavity. Panel C: A schematic showing how the subunit interfaces are stabilized by hydrophobic interactions, salt bridges, and hydrogen bonds, with specific interacting residues labeled at one interface.</image>
