Four Levels of Protein Structure

Protein structure is described at four levels: the primary structure is the sequence of amino acids, the secondary structure is the local folding into α-helices and β-sheets, the tertiary structure is the overall three-dimensional shape of one polypeptide, and the quaternary structure is the arrangement of several polypeptide subunits. In brief: an α-helix has 3.6 residues per turn and rises 0.15 nm per residue, so a helix of 100 residues is 15 nm long, and hemoglobin consists of four subunits (α₂β₂), each with one heme group.

The function of a protein depends on its three-dimensional shape, and the shape is fixed by the amino acid sequence. A change of one amino acid can alter the shape and cause disease. This article describes the four levels of structure with the forces that hold them together in a table, gives examples of each, shows calculations of helix dimensions and protein masses, and explains how the structure is determined and why the folding problem is so hard.

What is the primary structure?

The primary structure is the linear sequence of amino acids joined by peptide bonds, from the N-terminus to the C-terminus. It is determined by the sequence of the gene. A protein with n amino acids has 20ⁿ possible sequences, but only specific ones are useful. Even a tiny change can have big effects: in sickle-cell anemia, the replacement of one glutamic acid by valine at position 6 of the 146 amino acids in the β chain of hemoglobin makes the molecules stick together and deforms the red blood cells. The primary structure also includes the covalent disulfide bonds between cysteine residues. Insulin has 51 amino acids in two chains (A with 21 and B with 30), linked by two interchain disulfide bonds, and has one more within the A chain. All the higher levels of structure follow from the primary structure.

What is the secondary structure?

The secondary structure is the regular local arrangement of the backbone, stabilized by hydrogen bonds between the backbone C=O and N–H groups (not involving the side chains):

• α-Helix: a right-handed coil in which the C=O of residue n is hydrogen-bonded to the N–H of residue n + 4. There are 3.6 residues per turn, the rise is 0.15 nm per residue and the pitch 0.54 nm per turn. The side chains point outward. Keratin in hair and nails and myoglobin are rich in α-helices.
• β-Sheet: extended strands (rise of 0.33–0.35 nm per residue) lying side by side and linked by hydrogen bonds between strands. They can be parallel or antiparallel. Silk fibroin consists mainly of β-sheets, which is why silk is strong and not stretchable.
• Turns and loops: short regions that reverse the direction of the chain, often containing proline and glycine.

The permitted combinations of the angles φ and ψ were shown to be limited in the Ramachandran plot, as a consequence of the steric clashes between atoms.

What is the tertiary structure?

The tertiary structure is the complete three-dimensional fold of a single polypeptide chain, in which helices, sheets and loops are packed together into a compact shape. It is held together by interactions between the side chains:

• Hydrophobic effect: nonpolar side chains cluster in the interior, away from water. This is the main driving force of folding.
• Hydrogen bonds between polar side chains and with the backbone.
• Ionic bonds (salt bridges) between positively and negatively charged side chains, such as Lys⁺ and Glu⁻.
• Disulfide bonds (covalent) between two cysteine residues, common in secreted proteins such as insulin and antibodies.
• Van der Waals forces between closely packed atoms.

The fold creates the functional sites: for example, the active site of an enzyme is formed by amino acids that may be far apart in the sequence but close together in space. Globular proteins (enzymes, myoglobin) are compact and water-soluble, and fibrous proteins (collagen, keratin) are elongated structural proteins.

What is the quaternary structure?

Many proteins consist of two or more polypeptide chains (subunits), and the quaternary structure is the way in which they are assembled, held together by the same non-covalent forces (and sometimes disulfides). Hemoglobin is a tetramer (α₂β₂) of two α chains and two β chains, each with a heme group that binds one O₂ molecule, and a total molar mass of about 64,500 g/mol. Binding of oxygen to one subunit changes the shape of the others, and makes binding easier (cooperativity), giving the S-shaped oxygen binding curve. Not all proteins have a quaternary structure: myoglobin has a single chain. Other examples are antibodies (four chains), DNA polymerase and the proteasome.

Summary of the four levels

How do proteins fold?

Christian Anfinsen showed in the 1950s and 1960s that the enzyme ribonuclease A, after being completely unfolded with urea and a reducing agent, folds back into the active form when the denaturing agents are removed. This showed that the information for the 3D structure is contained in the amino acid sequence. Yet folding cannot happen by a random search: a chain of 100 residues with 3 possible conformations per residue would have 3¹⁰⁰ = 5 × 10⁴⁷ conformations, and even at 10¹³ trials per second, it would need about 10²⁷ years (Levinthal’s paradox). In reality, proteins fold within milliseconds to seconds by following a funnel-shaped energy landscape, in which the formation of local structures guides the folding. In the cell, chaperone proteins help to prevent aggregation. Misfolding is involved in diseases such as Alzheimer’s disease, Parkinson’s disease and prion diseases. The prediction of the structure from the sequence has been a challenge for decades, and was advanced greatly by the AlphaFold program in 2020–2021.

Worked examples

Example 1: length of an α-helix

A helix with 100 residues has a length of 100 × 0.15 nm = 15 nm, and has 100 / 3.6 = 27.8 turns, so 27.8 × 0.54 = 15.0 nm, which agrees. A fully extended chain (β-strand) of 100 residues would be about 100 × 0.34 = 34 nm long.

Example 2: the mass of a protein

A protein has 450 amino acids. The average residue mass is about 110 g/mol, so M ≈ 450 × 110 = 49,500 g/mol, or 49.5 kDa. Hemoglobin: 4 chains of about 141 and 146 residues: 2 × 141 + 2 × 146 = 574 residues, 574 × 110 = 63,100 g/mol, and the actual mass of 64,500 includes the four heme groups (616 g/mol each, 2464 g/mol in total).

Example 3: the number of disulfide bonds

Insulin has 6 cysteine residues (A6, A7, A11, A20, B7 and B19). They form 3 disulfide bonds. Each disulfide bond forms by the oxidation of two –SH groups: 2 RSH → RS–SR + 2H⁺ + 2e⁻.

Example 4: which level is affected?

• Mutation of one amino acid: primary.
• Heating that breaks the hydrogen bonds in helices: secondary (and higher).
• Reduction of disulfide bonds with mercaptoethanol: tertiary (and quaternary where the bonds are between chains).
• Dissociation of hemoglobin into subunits at low pH: quaternary.

How is protein structure determined?

• X-ray crystallography: the diffraction of X-rays by protein crystals gives an electron density map. Most structures in the Protein Data Bank were found this way.
• NMR spectroscopy: gives the structure of small and medium-sized proteins in solution.
• Cryo-electron microscopy: images of frozen molecules, now able to resolve large complexes to near-atomic detail.
• Mass spectrometry and Edman degradation: for the sequence.

Who discovered protein structure?

Frederick Sanger determined the first protein sequence (insulin) in 1955. Linus Pauling and Robert Corey proposed the α-helix and β-sheet in 1951, from model building and the planarity of the peptide bond, before any protein structure had been found by crystallography. John Kendrew solved the structure of myoglobin in 1958, and Max Perutz that of hemoglobin in 1959–1960, for which they shared the Nobel Prize in Chemistry in 1962. Anfinsen received the Nobel Prize in Chemistry in 1972 for his work on protein folding.

Which mistakes are common?

• Mixing up the levels: secondary structure uses the backbone only, tertiary involves the side chains.
• Thinking that all proteins have quaternary structure: only those with several chains.
• Saying that hydrogen bonds hold the primary structure together: peptide bonds are covalent.
• Forgetting that one amino acid can change the function: as in sickle-cell hemoglobin.
• Believing that disulfide bonds are the main force in all proteins: the hydrophobic effect usually dominates.