The intramolecular interactions discussed above stabilize the final folded structure of a protein. However, knowledge of the end states, N and U, tells us nothing of the path taken between them. Proteins fold on the time scale of microseconds to hundreds of seconds. It is impossible to sample all possible conformations during this time and it is clear that there is a preferred order of events leading to the final tertiary fold. Determining this order of events is an area of active inquiry. The questions that experimentalists are attempting to answer are "Do autonomously folding substructures nucleate the folding of other regions of the protein?" or ‘ ‘Do neighboring substructures fold and then collide to make the tertiary structure?" There is experimental evidence that hydrophobic amino acid residues collapse into a ‘ ‘hydrophobic core" and then the secondary structural units form around the core. It is likely that a combination of these scenarios leads to a correctly folded protein.
It is clear that the kinetics of protein folding is protein dependent. Some fold in a distinctly cooperative fashion, such that one can detect only the unfolded and native end states, being two-state in a kinetic as well as equilibrium sense. This is equivalent to saying that there is a single rate-limiting step, and intermediate species are not populated. Alternatively, some proteins fold by populating one or more distinct intermediate species. Thus, formation of the intermediate species is fast, often formed in the dead-time of the instrument, and formation of the native species from the intermediate is relatively slow and easily monitored experimentally. It has been shown that this slow phase in some cases may be due to proline isomerization.5
