Protein Folding Intermediates: Native-State Hydrogen Exchange

Abstract

The hydrogen exchange behavior of native cytochrome c in low concentrations of de-naturant reveals a sequence of metastable, partially unfolded forms that occupy free energy levels reaching up to the fully unfolded state. The step from one form to another is accomplished by the unfolding of one or more cooperative units of structure. The cooperative units are entire omega loops or mutually stabilizing pairs of whole helices and loops. The partially unfolded forms detected by hydrogen exchange appear to represent the major intermediates in the reversible, dynamic unfolding reactions that occur even at native conditions and thus may define the major pathway for cytochrome c folding.Under native conditions, a small fraction of any population of protein molecules occupies each possible higher energy, partially unfolded state, including even the fully unfolded state, as described by the Boltzmann distribution. The study of these partially unfolded forms (intermediates) may illuminate the fundamental cooperative nature of protein structure and define the unfolding and refolding pathways of a protein even though the intermediates are normally invisible to measurement. The energy levels and therefore the occupation of these conformationally excited states can be manipulated by denaturants and temperature. Hydrogen exchange experiments can then determine the hydrogens exposed in each higher energy form, their rates of exchange with solvent, and their sensitivity to the perturbant. From this we can infer, respectively, the structure, the free energy, and the surface exposure of each protein form.Results for cytochrome c reveal a small sequence of distinct partially unfolded forms with progressively increasing free energy and degree of unfolding. These appear to represent the major intermediates in the unfolding and refolding pathways of cytochrome c. Hydrogen exchange theoryExchangeable amide hydrogens (NH) that are involved in hydrogen-bonded structure can exchange with solvent hydrogens only when they are transiently exposed to solvent in some kind of closed to open reaction (1-3), as indicated in Eq. 1.(1)In the almost universally observed limiting case, referred to as EX2 (for bimolecular exchange) (1), the structural opening reaction enters the rate expression as a pre-equilibrium step. The exchange rate of any hydrogen, k ex , is then determined by its chemical exchange rate in the open form, k ch , multiplied by the equilibrium opening constant, K op (Eq. 2).

Determinants of protein hydrogen exchange studied in equine cytochrome c

“…The more buried NHs exhibit additional slowing. All these hydrogens exchange by way of local fluctuations except for Leu68NH which exchanges through a large subglobal unfolding (Bai et al, 1995). Helical periodicity of HX rates in amphipathic helices has been noted in other proteins (Goodman & Kim, 1991;Gooley et al, 1992;Radford et al, 1992;Pedersen et al, 1993;Orban et al, 1995).…”

“…The correlation plots in Figures 3-5 distinguish hydrogens that exchange by way of large structural unfolding reactions and local fluctuations. This distinction was made on the basis of the dependence of HX rate on guanidinium chloride concentration (Bai et al, 1995), which relates to the amount of surface newly exposed in the transient structural distortion that allows exchange to occur.…”

“…Local openings can be recognized by their near-zero dependence on added denaturant (rn value; Bai et al, 1995), signifying little new surface exposure in the exchange competent conformation. Also the observation that neighboring NHs show very different protection factors (Fig.…”

“…Large but still subglobal unfoldings have recently been shown to determine the exchange of some hydrogens in cyt c (Bai et al, 1995;Bai & Englander, 1996), ribonuclease H (Chamberlain et al, 1996), and a hyperthermophilic rubredoxin (Hiller et al, 1997). Exchange by way of large unfolding reactions can be recognized by the sharp dependence of rate on denaturant concentration, which relates to surface exposure in the unfolding reaction.…”

Protein folding: Then and now

“…The discovery of a class of simple, single-domain proteins which fold via two-state kinetics without any detectable intermediates in the early 1990s [6,7], the development of experimental techniques with improved spatial/temporal resolution [8][9][10][11][12][13], and the application of computer simulations using simplified lattice and off-lattice models [14,15] greatly enhanced our understanding of various aspects of the protein folding problem. Based on the nucleation theory [16][17][18], one of the early proposed mechanisms for protein folding, the nucleation-condensation model was formulated [19][20][21].…”