Experimental Identification of Downhill Protein Folding

Abstract

Theory predicts the existence of barrierless protein folding. Without barriers, folding should be noncooperative and the degree of native structure should be coupled to overall protein stability. We investigated the thermal unfolding of the peripheral subunit binding domain from Escherichia coli's 2-oxoglutarate dehydrogenase multienzyme complex (termed BBL) with a combination of spectroscopic techniques and calorimetry. Each technique probed a different feature of protein structure. BBL has a defined three-dimensional structure at low temperatures. However, each technique showed a distinct unfolding transition. Global analysis with a statistical mechanical model identified BBL as a downhill-folding protein. Because of BBL's biological function, we propose that downhill folders may be molecular rheostats, in which effects could be modulated by altering the distribution of an ensemble of structures.

Dynamics, Energetics, and Structure in Protein Folding

“…more disorder). Under this one-state scenario, the unfolding transition is structurally continuous and the conformational ensemble populated at each condition corresponds to a different stage of the folding reaction (67). The experiments and analyses described above demonstrate that global downhill and two-state-like folding constitute the two extremes of a continuous folding scale in which barriers heights range from significantly high (~15 RT) to non-existing.…”

“…The same oscillatory behavior can be exploited mechanically as a recoiling mechanism. Indeed, the molecular rheostat idea was put forward as an explanation for the complex functional roles that the small global downhill folder BBL plays in the oxo-glutarate dehydrogenase multienzyme complex (67). Other functionalities could be easily imagined: building a single molecule instantaneous sensor simply requires coupling the binding of the molecule to be detected to the folding of a one-state domain so that increased ligand concentrations induce a continuous change in conformation (and thus a gradual response).…”

Single‐Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics

“…Generally, protein folding occurs thermodynamically as a first-order transition, 29 although the recent experimental realization of downhill folding (see below) indicates that this does not always need to be the case. 30,31 Because of a limited set of model proteins available then, it was believed that each protein possesses a unique folding pathway from the unfolded to the folded protein, involving a discrete number of intermediate structures (the "old view" of protein folding). [32][33][34][35] With the discovery of so-called two-state folders (proteins that fold without intermediates) more than a decade ago, 36-38 this deterministic view of the folding process has changed radically.…”

A one-dimensional free energy surface does not account for two-probe folding kinetics of protein α3D

“…3,4 For a single activation barrier, such models predict exponential two-state folding kinetics, in which all thermodynamic and kinetic observables switch from unfolded reactant to folded product in a synchronized manner. 1 During the past decade, it has become recognized theoretically, 5 computationally, [6][7][8][9] and experimentally [10][11][12][13][14][15] that small proteins could fold much faster yet, approaching the downhill folding limit where the activation barrier becomes comparable to k B T. 16,17 Such efficiency is realized when short-range and long-range interactions within the protein and between protein and solvent harmonize en route to the native state ͑"consistency" 18 and "minimal frustration" 19 in the statistical mechanical literature͒. Proteins can also be poised at the transition state to fold downhill from there.…”