Dynamics of one-state downhill protein folding

Fig. 5.Free energy profiles for experimental reaction coordinates. (A) Free energy profiles as a function of the total number of helical residues at approximately every 14 K from 274 K (blue) to 344 K (yellow) for the P22 subdomain (Left) and αtα (Right). (B) The free energy as function of the folded probability for each individual 13 C-labeled stretch at two temperatures. The colors correspond to the label color scheme in Fig. 2. The apparent noise in some of the plots is due to the limited number of configurations for certain values of P, as stretches as short as two peptide bonds are considered.

mentioning

confidence: 99%

Sequence, structure, and cooperativity in folding of elementary protein structural motifs

Lai

Kubelka

2015

“…The folding time at 0.2 M GdnHCl, 27 ms, is ∼1350τ. This is consistent with the estimate of Zwanzig et al of a biased search in which the mean first-passage time of folding is τ f ≅ðτ∕NÞð1 þ KÞ N , where N is the chain length and K ∼ 0.2 is the equilibrium constant of forming an incorrect contact versus a correct contact, and suggests that the energetic cost of an incorrect contact is ∼2-3 k B T. Intriguingly, the relaxation of BBL at neutral pH and 300 K is very close to the reconfiguration time of protein L (24). Because BBL has little or no barrier between the folded and unfolded states, this time should be dominated by the conformational search time.…”

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...

“…Moreover, the free energy barriers separating folded and unfolded states for HP35(His27) have been estimated by both calorimetric and kinetic criteria to be very low (<2 kcal∕mol) (43); thus there is the possibility that the barrier disappears in the faster-folding mutant. In this so-called downhill scenario (44), we would expect to observe probe-dependent kinetics (45,46). On the other hand, if the kinetic properties are independent of the probe, then we can validate the use of fluorescence as a probe of global folding A UV laser pulse excites tryptophan to its lowest excited singlet state that undergoes intersystem crossing in a few nanoseconds to the triplet state, which then lives for up to 100 μs.…”

mentioning

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