There are 4 levels of protein structures:

• Primary: the amino acid sequence
• Secondary: local conformation of main-chain atoms (F and Y angles), how the amino acids in sequence fold up locally
• Tertiary: 3D folding or arrangement of the secondary structural elements and connecting loops in space.
• Quaternary: 3D arrangement of multiple subunits, each with a tertiary structure and each a unique gene product.

Primary Structure

A primary structure is a sequence of amino acids that are chemically bound together covalently. The amino acid sequence is responsible for the unique characteristics of every protein. Peptides are read from the amino terminus to the carboxyl terminal.

Amino acids bond together by peptide bonds. Peptide bonds are formed between the amino and carboxyl terminals and result in the release of a water molecule. Peptide bonds are usually formed in trans formation to prevent crowding. Gly is an important exception to this rule. The six atoms on the peptide group are co-planar.

The peptide bond is rigid and planar. Therefore, the polypeptide chain can only rotate about the bonds formed by C-α. These bonds have been termed the Phi φ and Psi ψ angles. The rotational freedom about φ and ψ angles is limited by steric hindrance between the side chains of the residues and the peptide backbone. Consequently, the possible conformations of a given polypeptide chain are quite limited.

• φ - C_α-N
• ψ - C_α-C'

cis & trans isomers

Dihedral angles

Dihedral angles are defined by 3 vectors, 4 atoms:

• φ - around Ca-N, 4 atoms C(i-1), N(i), Ca(i), C(i)
• ψ - around Ca-C, 4 atoms N(I), Ca(i), C(i), N(I+1)

Look along the appropriate bond, phi or psi "from the N terminal end".
A rotation of the bonds/atoms connected to the further end (C terminal end) in a "clockwise" sense is a positive rotation.
An anticlockwise rotation is negative

Peptide bond planes can rotate relative to each other:

• Hindered position: φ = 0, ψ = 0
• Fully extended position: φ = 180, ψ = 0

Ramachandran Plot

A Ramachandran Plot is a plot of φ vs. ψ angles. It maps the entire conformational space of a polypeptide and illuminates the allowed and disallowed conformations. Different amino acids have different preferences of φ-ψ angles.

Some key exceptions to these conformational limitations can be attributed to glycine and proline. The single H side chain of Glycine greatly reduces steric hindrance and expands the possible conformational space. The cyclic bond present in proline reduces the conformational space.

The nature of protein sequence and composition reflects its function. Membrane proteins have more hydrophobic residues. Homologous proteins often have similar sequences. Sequence similarity often implies similar secondary and tertiary structures.

Secondary Structure

The secondary structure refers to certain repetitive conformations in short sections of the peptide backbone. It can be thought of as the local conformation of the polypeptide chain, independent of the rest of the protein. The limitations imposed by peptide bonds and hydrogen bonding considerations dictate the secondary structure.

Some of the more commonly occuring secondary structures are:

• α-helix
• β-sheet
• turns
• random coils

φ and ψ conformations are specific and repetitive in α helices and β sheets. Conformations can be random in coils and loops.

α Helix

A helix is created by the curving of a polypeptide chain. The chain can coil to the right or the left. Almost all helices coil to the right. An α helix has 3.6 residues per turn. The structure is stabilized by N-H and C=O or the peptide group. Other helices such as π helix which has 4.4 residues per turn have and 3₁₀ helix which has 3 residues per turn have been observed in nature. However, such helices are rare.

R groups extend radially from the α helix core. The choice of residues extending from the helix make the helix polar, hydrophobic or amphipathic.

Different amino-acid sequences have different propensities for forming α helical structure. Methionine, alanine, leucine, glutamate, and lysine "MALEK" all have especially high helix-forming propensities. Proline tends to break or kink helices because it cannot donate an amide hydrogen bond (having no amide hydrogen), and because its sidechain interferes sterically; its ring structure also restricts its backbone. However, proline is often seen as the first residue of a helix, presumably due to its structural rigidity. At the other extreme, glycine also tends to disrupt helices because its high conformational flexibility makes it entropically expensive to adopt the relatively constrained α helical structure.

β Sheets

Unlike helices, β sheets are formed by hydrogen bonds between adjacent polypeptide chains rather than within a single chain. Sections of the polypeptide chain participating in the sheet are called β strands. β strands are formed by rotating φ and ψ approximately 180 degrees with respect to each other. Thus the peptide chains are fully extended and pleated because the adjacent peptides cannot be coplanar. β sheets are stabilized by interchaining hydrogen bonds between N-H and C=O.

β sheets can be either parallel or antiparallel. In a parallel configuration, both strands are lined up in the same direction e.g. C-terminal to N-terminal. In antiparallel configuration, one strand is lined N-terminal to C-terminal while the other is lined C-terminal to N-terminal. Parallel configuration does not have optimal hydrogen bond formation. Consequently, parallel configuration is less stable than antiparallel configuration. Mixed β sheets have both parallel and antiparallel configurations.

Secondary Structural Elements

Although, α helix and β sheets are the most predominant secondary structures, several irregular structures such as turns, loops, and coil are found in nature. Loops are usually present at the surface of the protein, often transitions between regular structures. Loops often act as an active site, e.g. on antibodies. Structurally speaking, turns and loops allow compaction of the protein.

Turns occur when there is a reversal in orientation of the main chain. They are stabilized by hydrogen bonds bridging across the interior of the turn. Loops are located on the surface of the protein. They connect two antiparallel strands.

Amino acids in α helices and β sheets show different geometries as can be seen in the ramachandran plot.

Right-handed α helix:

• φ –57°
• ψ –47°

Left-handed α helix:

• φ 60°
• ψ 60°

β strand

• φ -125°
• ψ 125°

Tertiary Structure

Tertiary structure is the 3D fold of the polypeptide structure in space. Motifs are limited number of secondary structure elements combined into simple folds. Domains are several motifs packed in a specific, compact arrangement that in many cases can fold as an independent unit. Large proteins consist of multiple domains connected by flexible segments of the peptide chain

Some general tertiary structures include:

• Hydrophobic residues collapse to form core of structure
• Hydrophilic residues are found on the surface
• Protein core is tightly packed
• Flexible regions are found on the surface

The tertiary structure is stabilized by weak noncovalent bonds such as van der waals forces, hydrophobic interactions, hydrogen bonds and salt bridges. Stronger convalent forces such as disulfide bonds and metal coordination also contribute to the overall stability.

Domains and Motifs

Domains are compact sections of the protein that represent structurally and functionally independent regions of a protein. This is to say that a domain is a subsection of the protein which would maintain its characteristic structure, even if separated from the overall protein. Motifs are substructures formed form a few secondary structures. Motifs are usually not structurally independent. They can be considered to be the minimal functional units of a protein. e.g.

• helix-loop-helix
• helix-strand-helix
• strand-loop-strand

Quaternary Structure

Quaternary structure is the association of multiple subunits (identical or different), each with a tertiary structure and each a unique gene product. Subunits are held together by many weak, noncovalent interactions (hydrophobic, electrostatic). Symmetry controls both structure and function of a quaternary structure.