B1.2 Proteins

Two of the four groups on the α carbon, the amine and carboxyl groups, are used up in forming peptide bonds, and the third is a hydrogen atom. So the properties of a polypeptide come almost entirely from its R groups, which differ greatly in chemistry, size and shape. This chemical diversity is the basis of the immense diversity of protein form and function.

R groups also differ in size, from a single hydrogen atom (glycine) to large ring structures, and some contain sulfur. Cysteine’s –SH group is special because two cysteines can be joined by a covalent disulfide bond.

Because each position in a chain can hold any of these, the R groups of a polypeptide decide whether its parts are attracted to water or to each other, where charges sit, and what other molecules it can bind. That is how one kind of building block produces enzymes, hormones, antibodies, channels and structural fibres.

Protein conformation is described at four levels. The first two are about the chain and its backbone.

Primary structure determines conformation

The primary structure is the number and sequence of amino acids in the polypeptide. As a ribosome adds amino acids, from the N-terminus to the C-terminus, the chain begins to fold, guided by the chemical properties of the R groups in their exact positions. Every molecule with the same sequence folds into the same shape, so proteins have precise, predictable and repeatable conformations despite their complexity.

Evidence:

• In the 1950s and 1960s Christian Anfinsen unfolded the enzyme ribonuclease with chemicals that also broke its disulfide bonds. When the chemicals were removed slowly, it refolded and became active again: the information for the shape is in the sequence.
• A single change can matter. In sickle-cell haemoglobin, glutamic acid (hydrophilic) is replaced by valine (hydrophobic) at position 6 of the β chain, and the molecules stick together when oxygen is low.
• Programs such as AlphaFold now predict the 3D structure of many proteins from their sequence alone.

Some proteins are helped to fold by chaperone proteins, but the final shape is still set by the sequence.

Secondary structure: coiling and pleating

The backbone of a polypeptide is a repeating –N–C–C– chain. Almost every N (not proline’s, which is bonded to its R group instead) carries a hydrogen (N–H) and every second C a double-bonded oxygen (C=O). These groups are slightly polar, so N–H of one part of the chain can hydrogen-bond to C=O of another. When the hydrogen bonds form at regular positions, the chain adopts a regular secondary structure:

• α-helix: the chain coils into a right-handed helix with 3.6 amino acids per turn. The C=O of each amino acid is hydrogen-bonded to the N–H of the amino acid four places further along, so all the bonds run roughly parallel to the axis, and the R groups point outwards.
• β-pleated sheet: two or more sections of chain lie side by side (running in the same or opposite directions). Hydrogen bonds form between C=O groups on one strand and N–H groups on the neighbouring strand. The sheet is pleated because the chain zig-zags at each α carbon, and the R groups point alternately above and below it.

Because the hydrogen bonds are at regular intervals, helices and sheets always have the same dimensions. Each hydrogen bond is weak, but there are very many of them. Regions without regular structure are often called loops or random coil.

The tertiary structure is the three-dimensional conformation of the whole polypeptide, produced as helices, sheets and loops fold up together. It is held by bonds and interactions between R groups, which may be far apart in the sequence but brought close together by folding:

How R groups become charged

Amine and carboxyl groups in R groups can gain or lose hydrogen ions:

• an amine group can accept H⁺: –NH₂ + H⁺ → –NH₃⁺ (positive);
• a carboxyl group can lose H⁺: –COOH → –COO⁻ + H⁺ (negative).

At the pH inside cells most of these groups are charged, so oppositely charged R groups attract and form ionic bonds. A change in pH adds or removes H⁺, changing these charges, which is why pH can break ionic bonds and denature proteins.

Disulfide bonds are covalent, so they lock parts of the chain together firmly. They are common in proteins secreted from cells, such as insulin and antibodies.

Where hydrophobic and hydrophilic amino acids sit decides both the shape of a protein and where in the cell it can be.

Water-soluble globular proteins

In a protein dissolved in water (in the cytoplasm, blood plasma or inside an organelle), the chain folds so that:

• hydrophobic (non-polar) R groups are clustered in the core, away from water, where hydrophobic interactions hold them together;
• hydrophilic (polar and charged) R groups are on the surface, forming hydrogen bonds with water, which keeps the protein soluble.

This arrangement is the lowest-energy one in water, so it stabilizes the tertiary structure. In the 3D model’s tertiary step, the yellow (hydrophobic) R groups end up packed between the helix and the sheet.

Integral membrane proteins

Proteins embedded in membranes have regions on their surface with hydrophobic amino acids, which are attracted to the hydrophobic core of the phospholipid bilayer and hold the protein in place. A common pattern is an α-helix of about 20 mostly non-polar amino acids, just long enough to span the bilayer. The parts that project into the water on either side, and any water-filled channel through the protein, are lined with hydrophilic amino acids. See B2.1 Membranes and membrane transport for the membrane itself.

Many proteins consist of a single polypeptide. Others are made of two or more polypeptides linked together: this is quaternary structure. The polypeptides (subunits) are held together by the same kinds of bonds as tertiary structure.

Some proteins also contain a non-polypeptide part, a prosthetic group. These are conjugated proteins; proteins made of polypeptide alone are non-conjugated.

Haemoglobin

Each of haemoglobin’s four polypeptides is folded around one haem group, a flat ring structure with an iron ion (Fe²⁺) at its centre. Each haem can bind one oxygen molecule, so one haemoglobin carries up to four O₂. The four subunits interact, so binding of oxygen to one changes the shape of the others and makes them bind oxygen more readily (see B3.1 Gas exchange).

Insulin is made as a single polypeptide that folds and forms its disulfide bonds; a middle section is then cut out, leaving the A and B chains linked. (Strictly, collagen carries a few sugar units on some of its amino acids, but it has no prosthetic group.)

Seeing single molecules: cryo-electron microscopy

Most protein structures were first worked out by X-ray crystallography, which needs the protein to form crystals. In cryogenic electron microscopy (cryo-EM), a solution of the protein is frozen so fast that the water forms glass-like (non-crystalline) ice, and the individual molecules are photographed with an electron microscope at very low temperature. Computers combine tens of thousands of images of molecules lying in different orientations into a 3D structure, often at near-atomic detail. Cryo-EM works for large complexes and membrane proteins that are hard to crystallize, and can show proteins bound to other molecules. Its developers shared the 2017 Nobel Prize in Chemistry, and it was used in 2020 to image the spike protein of the virus that causes COVID-19.

The shape of a protein suits it to its function.

Insulin: a globular hormone

Insulin is a small, compact protein secreted by β cells of the pancreas when blood glucose is high. Its hydrophilic surface lets it dissolve in blood plasma and travel round the body. Its precise, stable shape (helped by its disulfide bonds) fits a binding site on the insulin receptor in the plasma membranes of target cells such as liver, muscle and fat cells, triggering responses that lower blood glucose (see C2.1 Chemical signalling). Only molecules with exactly the right shape can bind — a general principle for globular proteins that bind other molecules, such as enzymes and their substrates (see C1.1 Enzymes and metabolism).

Collagen: a fibrous structural protein

Each collagen polypeptide has a repeating sequence in which every third amino acid is glycine, with proline and hydroxyproline also common. This sequence cannot form an α-helix; instead each chain forms a more extended, left-handed helix, and three chains wind round each other into a rope-like right-handed triple helix, about 300 nm long and 1.5 nm wide. Glycine, with the smallest R group, is the only amino acid that fits at the centre where the chains meet. Outside the cell, collagen molecules line up side by side and are covalently cross-linked into fibrils and fibres. Like a rope, collagen has very high tensile strength: it resists pulling forces without breaking. It is insoluble, so it stays in place in the extracellular matrix of skin, tendons, ligaments, cartilage, bone and blood vessel walls. It is the most abundant protein in mammals.