Protein structure

Why it matters

Proteins do most of the work in a cell: enzymes catalyse reactions, antibodies recognise invaders, transporters move ions, and structural proteins hold tissues together. What a protein does depends on its three-dimensional shape, and that shape is encoded in its amino acid sequence. Biochemists and biophysicists spend careers measuring, predicting and changing protein structure, so the four levels of structure are the vocabulary for everything else in this course.

Building blocks: amino acids and the peptide bond

Proteins are chains of the 20 standard amino acids. Each has the same backbone (an amino group, a central alpha carbon and a carboxyl group) and a different side chain (R group). Side chains are often grouped by chemistry:

• Nonpolar / hydrophobic: Ala, Val, Leu, Ile, Met, Phe, Trp, Pro. They tend to be buried inside the folded protein.
• Polar, uncharged: Ser, Thr, Asn, Gln, Tyr, Cys. They can form hydrogen bonds.
• Charged at pH 7: Asp and Glu (negative); Lys and Arg (positive); His is partly positive near neutral pH.
• Special cases: Gly is tiny and flexible; Pro locks the backbone and breaks helices; Cys can form disulfide bonds.

Amino acids join by peptide bonds, formed between the carboxyl of one and the amino group of the next. The peptide bond has partial double-bond character, so it is flat and rigid. The backbone can rotate only around two bonds per residue, described by the phi (φ) and psi (ψ) angles. A Ramachandran plot of φ against ψ shows which combinations are allowed without atoms clashing.

The four levels of structure

1. Primary structure is the amino acid sequence, written from the N-terminus to the C-terminus, such as MKTAYIAKQR.... It is held together by covalent peptide bonds and is read directly from the gene (or measured by mass spectrometry).

2. Secondary structure is the local folding of the backbone, stabilised by hydrogen bonds between backbone C=O and N–H groups:

• Alpha helix: a right-handed coil with 3.6 residues per turn; each C=O bonds to the N–H four residues along.
• Beta sheet: extended strands lying side by side, hydrogen bonded between strands, either parallel or antiparallel.
• Turns and loops connect them and are often where function happens.

3. Tertiary structure is the overall 3D fold of one polypeptide chain. It is driven mainly by the hydrophobic effect (nonpolar side chains cluster away from water) and stabilised by hydrogen bonds, ionic interactions (salt bridges), van der Waals contacts and, in many secreted proteins, disulfide bonds. Recurring folded units are called domains.

4. Quaternary structure is the arrangement of several chains (subunits) into one complex. Hemoglobin, for example, has four subunits (two alpha, two beta), and its subunits shift relative to each other when oxygen binds, which is the basis of cooperative binding.

Not every protein has quaternary structure, and some proteins or regions have no stable fold at all: intrinsically disordered regions are common and functionally important.

Worked example: reading a structure description

A database entry says: "Homodimer; each chain has an N-terminal Rossmann-fold domain (alternating alpha helices and parallel beta strands) and a C-terminal catalytic domain; one disulfide per chain."

• Quaternary: two identical chains (homodimer).
• Tertiary: two domains per chain.
• Secondary: helices and parallel beta strands in the first domain.
• Covalent extras: a disulfide bond within each chain.

Folding and stability

Anfinsen's experiments on ribonuclease showed that a denatured protein can refold on its own, so the sequence contains the information for the fold. In the cell, chaperones help many proteins fold and prevent aggregation. A folded protein is only marginally stable: the free energy difference between folded and unfolded states is often just 20–60 kJ/mol, the size of a few hydrogen bonds. Heat, extreme pH, detergents and chemical denaturants (urea, guanidinium) can unfold it. Misfolding and aggregation are linked to diseases such as Alzheimer's and Parkinson's.

Common mistakes

• Saying secondary structure comes from side-chain interactions. It comes from backbone hydrogen bonds; side chains mainly drive tertiary structure.
• Thinking disulfides hold most proteins together. Many intracellular proteins have none; the hydrophobic effect is the main driver of folding.
• Assuming one sequence means one rigid shape. Proteins breathe, flex and often change conformation when they bind something.

Recap

• Primary: sequence. Secondary: backbone hydrogen-bonded helices and sheets. Tertiary: whole-chain fold. Quaternary: multiple chains.
• The hydrophobic effect drives folding; many weak interactions stabilise it.
• Structure determines function, and small stability margins make proteins sensitive to their conditions.

Try it

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