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

Abstract

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...

A Compact Native 24-Residue Supersecondary Structure Derived from the Villin Headpiece Subdomain

“…In our first construct, we removed helix 1 from HP35wt and HP35stab, yielding HP24wt and HP24stab (Fig. 1 D) (26). We determined the helical content of the constructs from the analysis of far-UV CD spectra using DICHROWEB ( Fig.…”

“…The black lines are simulations of a two-state model (solid line) and three-state model (broken line) based on the parameters obtained from independent experiments. The data were taken from Zoldák et al (26). (D) CD spectra in the far-UV region.…”

“…By designing the villin headpiece subdomain HP35, Bi et al (25) found that two amino acid substitutions in helix 3 result in a dramatic stabilization of the whole protein, producing a hyperstable protein (~9 k B T vs.~4 k B T for HP35wt from bulk denaturation experiments). This N68A/K70M HP35 construct, named HP35stab (numbered according to the headpiece domain), showed higher stability against chemical denaturation as well as mechanical loads in single-molecule force experiments using optical tweezers (26). These experiments revealed a difference in folding cooperativity between HP35wt and HP35stab.…”

Understanding of the Physics of Biological Systems through the Use of Single-Molecule Magnetic Tweezers

“…[13][14][15] 특히, 광학 집게(optical tweezers)를 사용하여 전이 궤적을 측정 하고 생체분자의 구조 변화에 대해 생물물리학적으로 이해하려 는 최근 연구 결과에 대해 살펴보고자 한다. [23] Fig.…”

Hidden Markov Modeling with Detailed Balance and Its Application to Single Protein Folding

“…As the resolution of single-molecule measurements increases, complex reaction networks involving many states and multiple timescales have been studied. For example, latest high-resolution optical tweezers (OTs) are capable of detecting distance changes ranging from a few Å ngstroms to tens of microns in a timescale from a few microseconds to hours (17)(18)(19). As a result, equilibrium protein folding and unfolding has been observed among six states that significantly differ in their energies and lifetimes (11).…”