High-resolution x-ray crystal structures of the villin headpiece subdomain, an ultrafast folding protein

148

Equilibrium Fourier transform infrared (FTIR) and temperaturejump (T-jump) IR spectroscopic techniques were used to study the thermodynamics and kinetics of the unfolding and folding of the villin headpiece helical subdomain (HP36), a small three-helix protein. A double phenylalanine mutant (HP36 F47L, F51L) that destabilizes the hydrophobic core of this protein also was studied. The double mutant is less stable than wild type (WT) and has been shown to contain less residual secondary structure and tertiary contacts in its unfolded state. The relaxation kinetics after a T-jump perturbation were studied for both HP36 and HP36 F47L, F51L. Both proteins exhibited biphasic relaxation kinetics in response to a T-jump. The folding times for the WT (3.23 s at 60.2°C) and double phenylalanine mutant (3.01 s at 49.9°C) at the approximate midpoints of their thermal unfolding transitions were found to be similar. The folding time for the WT was determined to be 3.34 s at 49.9°C, similar to the folding time of the double phenylalanine mutant at that temperature. The double phenylalanine mutant, however, unfolds faster with an unfolding time of 3.01 s compared with 6.97 s for the WT at 49.9°C.protein folding ͉ Fourier transform IR ͉ temperature jump ͉ diffusion collision T he mechanism by which an unfolded polypeptide chain folds into its final native structure is still not fully understood, despite the obvious importance of the protein folding problem. In recent years there has been considerable interest in small single-domain proteins that fold quickly. These proteins provide attractive systems for computational and theoretical studies, and they are useful experimental models of the early steps in the assembly of more complicated folds. The helical subdomain derived from the villin headpiece, HP36, is a small three-helix structure found in the extreme C terminus of villin ( Fig. 1). Its modest size and helical structure suggest that it should fold rapidly, and this hypothesis has been independently confirmed by fluorescence-detected temperature-jump (T-jump) studies and NMR lineshape analysis (1-3). The helical subdomain is probably the smallest occurring natural sequence that has been shown to fold cooperatively. Its small size and very rapid folding have made it the focus of a large number of theoretical and computational studies (4-18).Residual structure and interactions in the unfolded state may play an important role in determining the rate of protein folding. Unfolded state structure might contribute to rapid folding by constraining and limiting the initial conformational search in the unfolded basin. Some models of folding postulate an important role for residual unfolded state structure. The diffusion collision model, for example, has been applied to rapidly folding helical proteins, and a key parameter in the model is the intrinsic stability of the individual microdomains (11,19,20). Although there have been a number of experimental studies of fast folding proteins, surprisingly little work has been reported that ...

Section: Resultsmentioning

confidence: 80%

Effect of modulating unfolded state structure on the folding kinetics of the villin headpiece subdomain

Brewer

Tang

et al. 2005

148

“…We validated that the Amber ff99SB*-ILDN (18,19,23) force field appears to be reasonably transferable across different protein classes (SI Text) and used it to investigate computationally the kinetics and thermodynamics of villin folding. In particular, we performed equilibrium MD simulations, at multiple temperatures, of the human villin C-terminal fragment (24), the Nle/Nle double mutant (3), and variants where we introduced an F10L mutant into either the wild-type protein or the Nle/Nle variant. Each simulation was run for at least 300 μs and contained between 30 and 150 folding and unfolding events ( Table 1).…”

Section: Resultsmentioning

confidence: 99%

Protein folding kinetics and thermodynamics from atomistic simulation

Piana

Lindorff‐Larsen

Shaw

2012

282

372

Advances in simulation techniques and computing hardware have created a substantial overlap between the timescales accessible to atomic-level simulations and those on which the fastest-folding proteins fold. Here we demonstrate, using simulations of four variants of the human villin headpiece, how simulations of spontaneous folding and unfolding can provide direct access to thermodynamic and kinetic quantities such as folding rates, free energies, folding enthalpies, heat capacities, Φ-values, and temperaturejump relaxation profiles. The quantitative comparison of simulation results with various forms of experimental data probing different aspects of the folding process can facilitate robust assessment of the accuracy of the calculations while providing a detailed structural interpretation for the experimental observations. In the example studied here, the analysis of folding rates, Φ-values, and folding pathways provides support for the notion that a norleucine double mutant of villin folds five times faster than the wild-type sequence, but following a slightly different pathway. This work showcases how computer simulation has now developed into a mature tool for the quantitative computational study of protein folding and dynamics that can provide a valuable complement to experimental techniques.Amber ff99SB*-ILDN | enthalpy | heat capacity | pre-exponential factor | transition path time P roteins are synthesized in the cell or in vitro as unstructured polypeptide chains that, in most cases, self-assemble into their functionally active three-dimensional shapes. This process, called protein folding, occurs on a broad range of timescales ranging from microseconds to seconds and higher. From a purely physical-chemical perspective, it should be possible in principle to characterize the folding mechanism of a given protein at atomistic resolution and to reconstruct its free-energy landscape, given only its primary sequence, through molecular dynamics (MD) simulations based on elementary physical principles. This direct approach has been rarely pursued because even the simplest systems representing a protein immersed in water consist of several thousand atoms, and simulating their behavior on the timescales typical of protein folding is computationally extremely demanding. The discovery and design of fast-folding proteins (1) significantly narrowed the timescale gap between simulations and experiments, making such simulations feasible, at least for the fastest-folding proteins.The C-terminal fragment of the villin headpiece [referred to in the remainder of this paper simply as "villin" (2)], one of the fastest-folding protein domains known (3), has proven to be an excellent target for folding simulations with physics-based force fields and an atomistically detailed representation of both the solute and the surrounding solvent (4-7). Until recently, the length of such simulations was limited to a few microseconds-a timescale sufficient to capture, at best, a single folding event (8,9). With this limitation, it has bee...

“…Only residues that are in contact in the native structure are considered and are defined by a separation between alpha carbons of Յ0.8 nm in the native structure (PDB entry 1YRF) (48). In this double sequence, approximation contacts can occur if all residues in the sequence between the contacting residues are in a native conformation.…”

Section: [6]mentioning

confidence: 99%

Measuring internal friction of an ultrafast-folding protein

Cellmer

Henry²,

Hofrichter³

et al. 2008

Self Cite

153

193

Nanosecond laser T-jump was used to measure the viscosity dependence of the folding kinetics of the villin subdomain under conditions where the viscogen has no effect on its equilibrium properties. The dependence of the unfolding/refolding relaxation time on solvent viscosity indicates a major contribution to the dynamics from internal friction. The internal friction increases with increasing temperature, suggesting a shift in the transition state along the reaction coordinate toward the native state with more compact structures, and therefore, a smaller diffusion coefficient due to increased landscape roughness. Fitting the data with an Ising-like model yields a relatively small position dependence for the diffusion coefficient. This finding is consistent with the excellent correlation found between experimental and calculated folding rates based on free energy barrier heights using the same diffusion coefficient for every protein.funneled energy landscape ͉ Ising-like model ͉ Kramers ͉ polypeptide ͉ viscosity D espite the complexity of the protein folding process, the kinetics and mechanisms of folding can be usefully and accurately described by diffusion over barriers on a lowdimensional free-energy surface (1-5). For ultrafast-folding proteins, the barriers are small and the rates may be affected by the variation of the diffusion coefficient along the reaction coordinate. In addition to solvent friction, the diffusion coefficient is determined by internal friction, which reflects the ''roughness'' of the energy landscape that arises from drag because of intrachain interactions and escape from local minima on the energy surface (1, 4, 6, 7). Both theoretical studies (1,8) and simulations (4, 9, 10) indicate that the internal friction depends on position along the reaction coordinate, but there have been no experiments that address this important issue in the physics of protein folding. In this work, we obtain a quantitative measure of the contribution of internal friction to the dynamics of folding from experiments on the viscosity dependence of the kinetics for the ultrafast-folding villin subdomain. Fitting the data with an Ising-like theoretical model, moreover, yields information on the position dependence of the diffusion coefficient. Unlike all previous studies of the viscosity dependence of protein-folding kinetics (11-17), we carried out experiments under conditions where there is no effect of the viscogen on the equilibrium thermal unfolding, as was done in a previous study of ␣-helix and ␤-hairpin formation (18).For barriers Ͼϳ 3RT separating folded and unfolded states, Kramers theory (19) predicts that the relaxation rate, 1/ , is given by:where is the relaxation time, f and u are the folding and unfolding times, ⌬G f ‡ is the free energy barrier to folding, ⌬G u ‡ is the free energy barrier to unfolding, R is the gas constant, T is the absolute temperature, ( u ) 2 is the curvature in the unfolded free energy well, ( f ) 2 is the curvature in the folded free energy well, ( ‡ ) 2 is the curvature a...