Rational Design, Structural and Thermodynamic Characterization of a Hyperstable Variant of the Villin Headpiece Helical Subdomain

In this study we expand the accessible dynamic range of singlemolecule force spectroscopy by optical tweezers to the microsecond range by fast sampling. We are able to investigate a single molecule for up to 15 min and with 300-kHz bandwidth as the protein undergoes tens of millions of folding/unfolding transitions. Using equilibrium analysis and autocorrelation analysis of the time traces, the full energetics as well as real-time kinetics of the ultrafast folding of villin headpiece 35 and a stable asparagine 68 alanine/lysine 70 methionine variant can be measured directly. We also performed Brownian dynamics simulations of the response of the bead-DNA system to protein-folding fluctuations. All key features of the force-dependent deflection fluctuations could be reproduced: SD, skewness, and autocorrelation function. Our measurements reveal a difference in folding pathway and cooperativity between wild-type and stable variant of headpiece 35. Autocorrelation force spectroscopy pushes the time resolution of single-molecule force spectroscopy to ∼10 μs thus approaching the timescales accessible for all atom molecular dynamics simulations. P rotein folding is a spontaneous process where a linear polypeptide chain acquires a functional and highly complex 3D structure. Conformational folding can occur on timescales ranging from hours down to microseconds (1, 2). The study of small fast-folding proteins has provided key information for understanding fundamental aspects of protein-folding mechanisms. Recently, it has even become possible to simulate folding trajectories of small proteins in full atomic detail using molecular dynamics simulations up to the millisecond time range (3, 4). However, direct time-resolved experimental measurements of such ultrafast processes have largely been limited to a few ensemble methods like temperature-jump, continuous-flow experiments, and triplet-lifetime measurements.The C-terminal subdomain of the actin-binding protein villin (villin headpiece, HP35) has been used as a model system in a number of both experimental and simulation studies of protein folding at the speed limit (5-19). The folding kinetics of HP35 has been studied extensively mostly by T-jump and by tripletlifetime experiments (6-8, 11, 12, 16). Folding of the wild-type HP35 at 30°C occurs at 348 × 10 3 s −1 , and unfolding at 7.4 × 10 3 s −1 , which in combination yields a free energy of folding of ∼4 k B T (6). Before crossing the major folding barrier, HP35 undergoes a rapid local rearrangement on the nanosecond timescale, which results in stable contacts of tertiary structure. Triplettriplet-energy transfer (TTET) measurements have reported a native-state heterogeneity and an additional high-energy intermediate on the native-state side of the major unfolding barrier (16). A detailed structural picture of the locking-unlocking reaction of the native states as observed in TTET data were given by recent all-atom molecular dynamics simulations (5). Along this line, a variety of native conformations was found consiste...

Section: Significancesupporting

confidence: 87%

Section: Significancementioning

confidence: 99%

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Ultrafast folding kinetics and cooperativity of villin headpiece in single-molecule force spectroscopy

Žoldák

Stigler

Pelz

et al. 2013

“…S1). Herein, we use a numbering system corresponding to the full-length villin headpiece: the first subdomain residue is L42; our construct contains an additional Met at the N terminus (M41) that does not affect the structure or folding of the protein (19). The calculated pI of the domain is 9.4 and the net charge at pH 7 is +2, depending on the exact pK a of the N terminus.…”

Section: Resultsmentioning

confidence: 99%

Rational modification of protein stability by targeting surface sites leads to complicated results

Xiao¹,

Patsalo

Shan³

et al. 2013

Self Cite

The rational modification of protein stability is an important goal of protein design. Protein surface electrostatic interactions are not evolutionarily optimized for stability and are an attractive target for the rational redesign of proteins. We show that surface charge mutants can exert stabilizing effects in distinct and unanticipated ways, including ones that are not predicted by existing methods, even when only solvent-exposed sites are targeted. Individual mutation of three solvent-exposed lysines in the villin headpiece subdomain significantly stabilizes the protein, but the mechanism of stabilization is very different in each case. One mutation destabilizes native-state electrostatic interactions but has a larger destabilizing effect on the denatured state, a second removes the desolvation penalty paid by the charged residue, whereas the third introduces unanticipated native-state interactions but does not alter electrostatics. Our results show that even seemingly intuitive mutations can exert their effects through unforeseen and complex interactions.atomistic simulations | pH-dependent stability | protein pKa measurements | nuclear magnetic resonance | protein biophysics T he mutation of charged surface residues to enhance electrostatics is a popular approach in protein engineering (1). Target residues are often selected using estimates of the protein electrostatic potential (2). This approach relies on identifying residues that are involved in unfavorable electrostatic interactions, typically with residues of the same charge, or on identifying sites where new favorable electrostatic interactions can be introduced (3). Solvent-exposed charged residues are thought to not be involved in critical packing interactions in the native state, to not suffer large desolvation penalties upon protein folding, nor to make significant interactions in the denatured-state ensemble (DSE). Thus, residues to be targeted are typically chosen on the basis of calculations of protein native-state ensemble (NSE) electrostatics. Methods ranging from simple inspection of the protein surface, modified Tanford-Kirkwood approaches (3, 4) and Poisson-Boltzmann (PB) calculations have been used (5-7). Irrespective of the details, the general strategy is based on the assumption that surface electrostatic interactions can be modified without altering other native-state interactions and without altering DSE energetics. An attractive feature of these approaches is that they are computationally inexpensive and, unlike selectionbased methods or directed evolution, involve the generation of a limited number of mutants. Any increase in stability is generally assumed to result from modification of NSE electrostatics. We show that this approach leads to complicated and unanticipated results, even for very simple proteins.We use the villin headpiece subdomain HP36 as our model system. HP36 is a small three-helix protein that has become an extremely popular model system for experimental and computational studies of protein folding, owing to its ...

“…The ϵ-amino nitrogen of Lys24 is 6.1 Å from the nearest protonated nitrogen of the imidazole ring of His27 and the ϵ-amino nitrogen of Lys29 is 5.3 Å from the nearest charged amino atom of Arg14. Removal of the ϵ-amino nitrogen groups of Lys24 and Lys29 to form norleucines in the mutant called HP35 (Nle24,His27,Nle29) eliminates the repulsive interaction, thereby stabilizing the protein and increasing the folding rate (24,25).…”

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