Though the structures presented in crystallographic models of macromolecules appear to possess rock-like solidity, real proteins and nucleic acids are not particularly rigid. Most structural work to date has centred upon the native state of macromolecules, the most probable macromolecular form. But the native state of a molecule is merely its most abundant form, certainly not its only form. Thermodynamics requires that all other possible structural forms, however improbable, must also exist, albeit with representation corresponding to the factor exp( — Gi/RT) for each state of free energy Gi (see Moelwyn-Hughes, 1961), and one appreciates that each molecule within a population of molecules will in time explore the vast ensemble of possible structural states.

In order to solve the immensely difficult protein-folding problem, it will be necessary to characterize the barriers that slow folding and the intermediate structures that promote it. Although protein-folding intermediates are not accessible to the usual structural studies, hydrogen exchange (HX) methods have been able to detect and characterize intermediates in both kinetic and equilibrium modes--as transient kinetic folding intermediates on a subsecond time scale, as labile equilibrium molten globule intermediates under destabilizing conditions, and as infinitesimally populated intermediates in the high free-energy folding landscape under native conditions. Available results consistently indicate that protein-folding landscapes are dominated by a small number of discrete, metastable, native-like partially unfolded forms (PUFs). The PUFs appear to be produced, one from another, by the unfolding and refolding of the protein's intrinsically cooperative secondary structural elements, which can spontaneously create stepwise unfolding and refolding pathways. Kinetic experiments identify three kinds of barrier processes: (a) an initial intrinsic search-nucleation-collapse process that prepares the chain for intermediate formation by pinning it into a condensed coarsely native-like topology; (b) smaller search-dependent barriers that put the secondary structural units into place; and (c) optional error-dependent misfold-reorganization barriers that can cause slow folding, intermediate accumulation, and folding heterogeneity. These conclusions provide a coherent explanation for the grossly disparate folding behavior of different globular proteins in terms of distinct folding pathways.

Previous hydrogen exchange (HX) studies of the spontaneous reversible unfolding of Cytochrome c (Cyt c) under native conditions have led to the following conclusions. Native Cyt c (104 residues) is composed of five cooperative folding units, called foldons. The highenergy landscape is dominated by an energy ladder of partially folded forms that differ from each other by one cooperative foldon unit. The reversible equilibrium unfolding of native Cyt c steps up through these intermediate forms to the unfolded state in an energy-ordered sequence, one foldon unit at a time. To more directly study Cyt c intermediates and pathways during normal energetically downhill kinetic folding, the present work used HX pulse labeling analyzed by a fragment separation-mass spectrometry method. The results show that 95% or more of the Cyt c population folds by stepping down through the same set of foldon-dependent pathway intermediates as in energetically uphill equilibrium unfolding. These results add to growing evidence that proteins fold through a classical pathway sequence of native-like intermediates rather than through a vast number of undefinable intermediates and pathways. The present results also emphasize the condition-dependent nature of kinetic barriers, which, with less informative experimental methods (fluorescence, etc.), are often confused with variability in intermediates and pathways.protein folding | foldons | cytochrome c T he protein-folding problem, one of the oldest and most difficult in all of biophysics, lies at the heart of biology. Proteins must fold to their active native state when they are initially released from the ribosome and when they unfold and refold, over and over again, during their lifetime (1). It is one reaction that all organisms-animals, plants, and microbes-absolutely require. However, 55 y after Anfinsen et al. showed that proteins can fold all by themselves without outside help (2), no generally accepted folding model has emerged.Soon after Anfinsen's demonstration, Levinthal noted that sizeable proteins could not find their native state within any reasonable folding time by a random search through the vast conformational space available to them (3). However, proteins can fold in milliseconds. He concluded that proteins must fold through some programmed structure formation pathway, analogous to other biochemical pathways, although one had no idea how such a pathway might look or what could guide it. A different answer to the Levinthal paradox, focused on energy landscape considerations, points out that the search is not wholly random. Refolding proteins must naturally diffuse energetically downhill stepping through a landscape that contains all possible intermediates and pathways. In the absence of more detailed direction, structural search processes at the microscopic, near single-residue level must carry protein populations heterogeneously through all of these structural forms and different tracks, chosen perhaps by each protein's initial unfolded conformation (4-7).The discovery th...

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