Protein folded states are kinetic hubs

107

We have performed the first unbiased folding simulations of the GB1 hairpin in explicit solvent, using hundreds of microsecondlong molecular dynamics simulations (total time: 0.7 ms). Our simulations are initiated from two sets of structures. Starting from an equilibrium unfolded state, we obtain single-exponential folding kinetics with rate coefficients in good agreement (T ¼ 350 K) or within an order of magnitude (T ¼ 300 K) of the experimental values. However, simulations initiated from unfolded configurations lacking secondary structure result in biexponential kinetics with an additional fast nanosecond kinetic mode. This mode can strongly bias the folding rate estimated from the mean first passage time, when the trials are much shorter than the folding time. We find that the mechanism of the hairpin folding is insensitive to the details of the initial unfolded ensemble and is initiated by correct formation of the turn of the hairpin, followed by the formation of the native hydrogen bonds and hydrophobic contacts, consistent with experimental ϕ-value analysis. Subsequent native interactions can be formed either from the turn or from the hairpin termini, helping to explain an apparent discrepancy in experimental results. From our simulations, we also obtain the transition path durations, a critical parameter for single molecule experiments aiming to resolve events along folding pathways. The lengths of transition paths span a wide range, from 50 ps to 140 ns, at 300 K.protein folding | folding mechanism | transition path time

Section: Discussionmentioning

confidence: 99%

Microscopic events in β-hairpin folding from alternative unfolded ensembles

Best

Mittal²

2011

107

“…Much theoretical work has described protein folding as diffusion on an energy landscape, so a detailed understanding of the folding pathway requires knowledge of how unfolded states intercon- [GdnHCl] (M) (21,22). A set of experiments on the ultrafast folder BBL, a candidate for downhill folding, have observed the rate of intramolecular diffusion as a relaxation after T-jump.…”

Section: Discussionmentioning

confidence: 99%

Extremely slow intramolecular diffusion in unfolded protein L

Waldauer

Bakajin

Lapidus³

2010

105

A crucial parameter in many theories of protein folding is the rate of diffusion over the energy landscape. Using a microfluidic mixer we have observed the rate of intramolecular diffusion within the unfolded B1 domain of protein L before it folds. The diffusionlimited rate of intramolecular contact is about 20 times slower than the rate in 6 M GdnHCl, and because in these conditions the protein is also more compact, the intramolecular diffusion coefficient decreases 100-500 times. The dramatic slowdown in diffusion occurs within the 250 μs mixing time of the mixer, and there appears to be no further evolution of this rate before reaching the transition state of folding. We show that observed folding rates are well predicted by a Kramers model with a denaturant-dependent diffusion coefficient and speculate that this diffusion coefficient is a significant contribution to the observed rate of folding. microfluidic mixing | protein folding | unfolded state T he question of what determines the rate that a polypeptide chain finds the lowest energy native state has been a longstanding debate in the field of protein folding. Seminal work by Baker and coworkers a decade ago showed a remarkable correlation between the contact order of the native state and the folding rate (1). This correlation is particularly strong among two-state folders that have only one significant barrier between the folded and unfolded states. The observation led to much theoretical work using Go models to generate a folding landscape upon which the folding protein traversed with a certain rate of diffusion (2, 3). The concept of diffusion over a landscape is not new; Kramers showed the rate of crossing a 1-dimensional reaction barrier also depended on the viscosity of the system (4). However, neither type of model can directly determine the rate of diffusion on an energy surface.The search for the appropriate diffusion coefficient for these types of models led several groups to investigate the intramolecular diffusion time of small loops in random polypeptides (5-8). These peptides were typically very flexible and highly diffusive. The typical observed diffusion coefficient, D ∼ 10 6 cm 2 s −1 , is less than 10 times slower than the free diffusion of individual amino acids (5-8). Kubelka et al. used this diffusion coefficient to calculate the reconfiguration time of an unfolded protein to produce the well-known estimate of the protein folding "speed limit" of N∕100 μs (9). However, later work has shown that for real proteins in denaturant, the unfolded state compacts and D decreases as denaturant is reduced, but these studies were limited to conditions in which a detectable population of unfolded molecules is present in equilibrium (10, 11). In this work we present a unique measurement in which intramolecular diffusion of a folding protein is measured in a microfluidic mixer. This mixer allows measurement of intramolecular diffusion of the true unfolded state before the protein folds. We find that the diffusion coefficient of B1 domain of protein...

“…Bowman et al (29,52) used a Markov state model to analyze the trajectories of Ensign et al (27). A biexponential fit to the (blue) folded population vs. time plot in figure 6 of ref.…”

Section: Discussionmentioning

confidence: 99%

Making connections between ultrafast protein folding kinetics and molecular dynamics simulations

Cellmer

Buscaglia

Henry

et al. 2011

Determining the rate of forming the truly folded conformation of ultrafast folding proteins is an important issue for both experiments and simulations. The double-norleucine mutant of the 35-residue villin subdomain is the focus of recent computer simulations with atomistic molecular dynamics because it is currently the fastest folding protein. The folding kinetics of this protein have been measured in laser temperature-jump experiments using tryptophan fluorescence as a probe of overall folding. The conclusion from the simulations, however, is that the rate determined by fluorescence is significantly larger than the rate of overall folding. We have therefore employed an independent experimental method to determine the folding rate. The decay of the tryptophan triplet-state in photoselection experiments was used to monitor the change in the unfolded population for a sequence of the villin subdomain with one amino acid difference from that of the laser temperature-jump experiments, but with almost identical equilibrium properties. Folding times obtained in a two-state analysis of the results from the two methods at denaturant concentrations varying from 1.5-6.0 M guanidinium chloride are in excellent agreement, with an average difference of only 20%. Polynomial extrapolation of all the data to zero denaturant yields a folding time of 220 ðþ100, − 70Þ ns at 283 K, suggesting that under these conditions the barrier between folded and unfolded states has effectively disappeared-the so-called "downhill scenario." tryptophan triplet lifetime | villin headpiece subdomain | downhill protein folding | laser temperature jump