Protein folding and stability

Levels of structure

• Primary: the amino acid sequence, plus covalent features such as disulfides.
• Secondary: local backbone hydrogen-bonded patterns. In the right-handed \alpha-helix, each C=O hydrogen-bonds to the N–H four residues along (i \to i+4), with 3.6 residues per turn. \beta-sheets form from extended strands hydrogen-bonded side by side, parallel or antiparallel; turns reverse chain direction, often using Gly and Pro.
• Tertiary: the overall fold of one chain, typically helices and sheets packed to bury hydrophobic side chains.
• Quaternary: the assembly of several chains, such as the \alpha_2\beta_2 tetramer of haemoglobin.

The peptide bond is planar (partial C=N character) and usually trans, so backbone shape is set by two torsion angles, \phi and \psi, per residue. The Ramachandran plot shows that steric clashes allow only a few regions, corresponding to helix and sheet; glycine is unusually flexible and proline unusually restricted.

The thermodynamics of folding

The free energy of unfolding, \Delta G_{\text{unf}} = G_U - G_F, is typically only about 20–60 kJ mol^{-1}: a small difference between large opposing terms.

• The hydrophobic effect is the main driving force. Water around exposed non-polar groups is more ordered; burying them releases that water, an entropy gain for the solvent that outweighs the loss of chain conformational entropy.
• Hydrogen bonds within the protein contribute less than might be expected, because the unfolded chain already hydrogen-bonds to water. They are, however, essential for specificity: a buried unsatisfied donor or acceptor is very costly.
• Salt bridges between, for example, Lys and Glu side chains help mainly when partially buried; on the surface they are screened by water and ions.
• Van der Waals packing in the tightly packed core is substantial.
• Disulfide bonds between Cys residues, formed oxidatively mainly in extracellular proteins, stabilise the folded state by lowering the entropy of the unfolded chain.

How folding happens

Anfinsen showed that ribonuclease A, unfolded with urea and with its disulfides reduced, refolds spontaneously with full activity once the denaturant is removed: the sequence encodes the structure. Yet a random search through all conformations would take astronomically long (the Levinthal paradox). The resolution is a funnel-shaped energy landscape: many routes lead downhill, with local structure forming early and guiding the collapse. Small proteins often fold in a two-state way with a single transition state.

Stability and denaturation

Heat, extremes of pH, urea and guanidinium chloride unfold proteins. Following unfolding by CD or fluorescence against denaturant concentration gives \Delta G_{\text{unf}} by extrapolation to zero denaturant. In the crowded cell, chaperones such as the GroEL/GroES cage prevent aggregation by giving partly folded chains a protected space to fold.