The integration of atomic-resolution experimental and computational methods offers the potential for elucidating key aspects of protein folding that are not revealed by either approach alone. Here, we combine equilibrium NMR measurements of thermal unfolding and long molecular dynamics simulations to investigate the folding of gpW, a protein with two-state-like, fast folding dynamics and cooperative equilibrium unfolding behavior. Experiments and simulations expose a remarkably complex pattern of structural changes that occur at the atomic level and from which the detailed network of residue–residue couplings associated with cooperative folding emerges. Such thermodynamic residue–residue couplings appear to be linked to the order of mechanistically significant events that take place during the folding process. Our results on gpW indicate that the methods employed in this study are likely to prove broadly applicable to the fine analysis of folding mechanisms in fast folding proteins.

A one-state downhill protein folding process is barrierless at all conditions, resulting in gradual melting of native structure that permits resolving folding mechanisms step-by-step at atomic resolution. Experimental studies of one-state downhill folding have typically focused on the thermal denaturation of proteins that fold near the speed limit (ca. 10 6 s −1 ) at their unfolding temperature, thus being several orders of magnitude too fast for current single-molecule methods, such as single-molecule FRET. An important open question is whether one-state downhill folding kinetics can be slowed down to make them accessible to single-molecule approaches without turning the protein into a conventional activated folder. Here we address this question on the small helical protein BBL, a paradigm of one-state downhill thermal (un)folding. We decreased 200-fold the BBL folding-unfolding rate by combining chemical denaturation and low temperature, and carried out freediffusion single-molecule FRET experiments with 50-μs resolution and maximal photoprotection using a recently developed Troloxcysteamine cocktail. These experiments revealed a single conformational ensemble at all denaturing conditions. The chemical unfolding of BBL was then manifested by the gradual change of this unique ensemble, which shifts from high to low FRET efficiency and becomes broader at increasing denaturant. Furthermore, using detailed quantitative analysis, we could rule out the possibility that the BBL single-molecule data are produced by partly overlapping folded and unfolded peaks. Thus, our results demonstrate the onestate downhill folding regime at the single-molecule level and highlight that this folding scenario is not necessarily associated with ultrafast kinetics. P rotein folding is an ideal problem for single-molecule approaches because the simple collective behavior that is frequently observed in bulk experiments could hide an underlying complexity of myriads of microscopic folding pathways (1). Thus protein folding has been a major target for modern single-molecule experiments, including force-microscopy (2) and fluorescence (3). Among these, single-molecule FRET (SM-FRET) has the advantage of recapitulating the conventional bulk chemical unfolding experiments at the single-molecule level. SM-FRET methods have already made important contributions to protein folding, such as demonstrating the conversion between native and unfolded populations of two-state-like folding (4), resolving the chemical-denaturant-induced expansion of the unfolded state (5) and its nanosecond conformational dynamics (6), and setting upper bounds for folding transition-path times (7).Another important application is the characterization of the downhill folding scenario predicted by energy landscape theory (1). Downhill folding proteins have a maximal free-energy barrier (i.e., at the denaturation midpoint) below 3RT. (RT is thermal energy, where R is the gas constant and T is the temperature in Kelvin.) The barrier top is thus significantly populated, and ...

Direct evidence of superradiance from an organic semiconductor (quaterthiophene) whose molecules are arranged in a H aggregate fashion, is reported. Time resolved photoluminescence measurements show a linear correlation between the radiative lifetime (tau(rad)) of the purely electronic exciton recombination and the inverse of the number (N(C)) of the coherently emitting dipoles, i.e., tau(rad) proportional, variant 1/N(C). These data support the recently developed theoretical models describing the optical properties of H aggregates of rodlike molecules with a nonvanishing component of the perpendicular molecular transition dipole moment.

Protein folding research stalled for decades because conventional experiments indicated that proteins fold slowly and in single strokes, whereas theory predicted a complex interplay between dynamics and energetics resulting in myriad microscopic pathways. Ultrafast kinetic methods turned the field upside down by providing the means to probe fundamental aspects of folding, test theoretical predictions and benchmark simulations. Accordingly, experimentalists could measure the timescales for all relevant folding motions, determine the folding speed limit and confirm that folding barriers are entropic bottlenecks. Moreover, a catalogue of proteins that fold extremely fast (microseconds) could be identified. Such fast-folding proteins cross shallow free energy barriers or fold downhill, and thus unfold with minimal co-operativity (gradually). A new generation of thermodynamic methods has exploited this property to map folding landscapes, interaction networks and mechanisms at nearly atomic resolution. In parallel, modern molecular dynamics simulations have finally reached the timescales required to watch fast-folding proteins fold and unfold in silico. All of these findings have buttressed the fundamentals of protein folding predicted by theory, and are now offering the first glimpses at the underlying mechanisms. Fast folding appears to also have functional implications as recent results connect downhill folding with intrinsically disordered proteins, their complex binding modes and ability to moonlight. These connections suggest that the coupling between downhill (un)folding and binding enables such protein domains to operate analogically as conformational rheostats.