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A protein is synthesized as a polypeptide whose sequence constrains its possible interactions. Protein folding is the acquisition of a functional three-dimensional structure: local hydrogen bonds organize secondary structure, while packing, electrostatic interactions and burial of nonpolar groups through the hydrophobic effect stabilize the overall fold. Some proteins also form protein disulfide bonds. Folding reduces the chain's conformational entropy, but the relevant balance includes the entropy of the surrounding water; it is the total Gibbs free energy that is minimized at fixed pressure and temperature.

A folding funnel describes a biased but rugged energy landscape: increasingly native-like conformations become favorable, while barriers can trap intermediates. The chain therefore does not have to test every possible conformation in a uniform random search. Molecular chaperones can prevent protein aggregation and help escape kinetic traps; some proteins remain disordered or require partners, so a unique isolated compact fold is not universal.

The ribonuclease A refolding experiment connects this thermodynamic account to a measurement. Unfolding the enzyme with urea while reducing its disulfide bonds removes activity; suitable removal of the denaturant and restoration of disulfide bonds recover activity. Oxidizing while the chain remains unfolded instead produces incorrectly paired disulfides and little activity. Thus sequence, solvent conditions and the route past kinetic traps all matter; restoration of enzyme activity provides a functional test of folding.