Single-molecule FRET Study of Denaturant Induced Unfolding of RNase H

270

304

Transition paths are a uniquely single-molecule property not yet observed for any molecular process in solution. The duration of transition paths is the tiny fraction of the time in an equilibrium single-molecule trajectory when the process actually happens. Here, we report the determination of an upper bound for the transition path time for protein folding from photon-by-photon trajectories. FRET trajectories were measured on single molecules of the dyelabeled, 56-residue 2-state protein GB1, immobilized on a glass surface via a biotin-streptavidin-biotin linkage. Characterization of individual emitted photons by their wavelength, polarization, and absolute and relative time of arrival after picosecond excitation allowed the determination of distributions of FRET efficiencies, donor and acceptor lifetimes, steady state polarizations, and waiting times in the folded and unfolded states. Comparison with the results for freely diffusing molecules showed that immobilization has no detectable effect on the structure or dynamics of the unfolded protein and only a small effect on the folding/unfolding kinetics. Analysis of the photon-by-photon trajectories yields a transition path time <200 s, >10,000 times shorter than the mean waiting time in the unfolded state (the inverse of the folding rate coefficient). Szabo's theory for diffusive transition paths shows that this upper bound for the transition path time is consistent with previous estimates of the Kramers preexponential factor for the rate coefficient, and predicts that the transition path time is remarkably insensitive to the folding rate, with only a 2-fold difference for rate coefficients that differ by 10 5 -fold.A detailed description and understanding of mechanisms of protein folding has been one of the great challenges to biophysical science. The simplest system to study, and the one that has produced the most insights, is a protein exhibiting 2-state behavior (1-7). A 2-state protein has only 2-populations of molecules in equilibrium and at all times in kinetic experiments-folded and unfolded. In ensemble folding experiments kinetics are studied by rapidly changing solution conditions, e.g., the temperature or denaturant concentration, and monitoring the relaxation of the 2 populations to their new equilibrium ratio with probes such as fluorescence, circular dichroism or infrared spectroscopy. Single molecule kinetics, however, can be studied at equilibrium. As can be seen from the schematic of a trajectory in Fig. 1, the dynamical nature of equilibrium is dramatically demonstrated when observing Förster resonance energy transfer (FRET) in a single-molecule fluorescence experiment. There are fluctuations due to shot noise about a mean value in each state, interrupted by what appear to be instantaneous jumps in FRET efficiency signaling folding or unfolding. The residence or waiting times in each state are exponentially distributed, with the mean time in the unfolded and folded segments of the trajectories corresponding to the inverse of the folding and ...

Section: Discussionsupporting

confidence: 60%

Experimental determination of upper bound for transition path times in protein folding from single-molecule photon-by-photon trajectories

Chung

Louis²,

Eaton³

2009

270

304

“…(Left) Shown, for example, are SAXS profiles collected on protein L at equilibrium at 1 M (native conditions), at equilibrium at 4 M GuHCl (unfolding conditions), and transiently before folding on transfer to denaturant concentrations as low as 0.67 M; the near-superimposability of the scaled scattering data for the various unfolded states suggests that the unfolded chain does not change dimensions (to within experimental uncertainty of a few percent) before folding (4). (Right) The efficiency of smFRET observed across dye-labeled unfolded proteins, in contrast, invariably decreases significantly as the level of denaturant rises (6,7,16,19,20,23), an observation that has universally been interpreted in terms of an unfolded state that expands significantly at higher denaturant concentrations. Shown are equilibrium smFRET data collected on dye-labeled protein L as a function of GuHCl concentration (6); the large shift in the placement of the peak corresponding to unfolded molecules (indicated by the blue bar) suggests that the chain contracts significantly when transferred from high to low denaturant.…”

Section: Resultsmentioning

confidence: 99%

“…1). Moreover, this discrepancy seems to be nearly universal among single-domain proteins: whereas the results of at least a half-dozen SAXS studies on chemically unmodified, single-domain proteins fail to detect any experimentally significant evidence (at experimental precision of, typically, a few percent) of contraction (8)(9)(10)(11)(12)(13)(14)(15), all of the more than one-dozen smFRET studies of dye-labeled, singledomain proteins reported to date have been interpreted in terms of unfolded states that contract as the denaturant concentration is lowered to ∼1 M (2,6,7,(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26).…”

mentioning

confidence: 99%

Random coil negative control reproduces the discrepancy between scattering and FRET measurements of denatured protein dimensions

Watkins

Simon

Sosnick

et al. 2015

Small-angle scattering studies generally indicate that the dimensions of unfolded single-domain proteins are independent (to within experimental uncertainty of a few percent) of denaturant concentration. In contrast, single-molecule FRET (smFRET) studies invariably suggest that protein unfolded states contract significantly as the denaturant concentration falls from high (∼6 M) to low (∼1 M). Here, we explore this discrepancy by using PEG to perform a hitherto absent negative control. This uncharged, highly hydrophilic polymer has been shown by multiple independent techniques to behave as a random coil in water, suggesting that it is unlikely to expand further on the addition of denaturant. Consistent with this observation, small-angle neutron scattering indicates that the dimensions of PEG are not significantly altered by the presence of either guanidine hydrochloride or urea. smFRET measurements on a PEG construct modified with the most commonly used FRET dye pair, however, produce denaturant-dependent changes in transfer efficiency similar to those seen for a number of unfolded proteins. Given the vastly different chemistries of PEG and unfolded proteins and the significant evidence that dye-free PEG is well-described as a denaturant-independent random coil, this similarity raises questions regarding the interpretation of smFRET data in terms of the hydrogen bond-or hydrophobically driven contraction of the unfolded state at low denaturant.protein folding | SAXS | two state | statistical coil | Flory scaling R ecent years have seen a significant controversy arise regarding the behavior of the unfolded states of single-domain proteins in response to changing levels of chemical denaturant. That is, although small-angle X-ray scattering (SAXS) and single-molecule FRET (smFRET) results are all consistent with the argument that, at high levels of chemical denaturant (∼6 M or above), unfolded proteins adopt a swollen, self-avoiding ensemble well-approximated as an excluded volume random coil (1-3), the two approaches produce seemingly discrepant results for the dimensions of the unfolded states populated at lower (∼1 M) denaturant (4). For example, time-resolved SAXS studies of the unfolded state transiently populated when protein L is rapidly shifted from high guanidine hydrochloride (GuHCl) to low denaturant conditions produce no experimentally significant evidence for the compaction of this single-domain protein before folding (4, 5) [e.g., estimated radii of gyration of 25.1 ± 0.3 Å for the unfolded state at equilibrium in 7 M GuHCl and 24.9 ± 1.1 Å for the transient unfolded state populated at 0.67 M GuHCl; confidence intervals are SEs (4)]. In contrast, multiple smFRET studies conducted at equilibrium suggest that the unfolded state of dye-labeled protein L contracts by 20-40% over this same range of denaturant concentrations (6, 7) (Fig.

“…Recently, the collapse of unfolded molecules of the small cold shock protein CspTm at close to native conditions was discovered with this approach (14). This collapsed unfolded form also can be populated kinetically (15, 16) and has by now been found for a range of small proteins (16)(17)(18)(19)(20). It is unclear, however, whether this collapse is a nonspecific random heteropolymer collapse (21) or whether it is accompanied by the formation of specific structure.…”

mentioning

confidence: 93%

Mapping protein collapse with single-molecule fluorescence and kinetic synchrotron radiation circular dichroism spectroscopy

Hoffmann

Kane

Nettels

et al. 2007

217

338

We have used the combination of single-molecule Fö rster resonance energy transfer and kinetic synchrotron radiation circular dichroism experiments to probe the conformational ensemble of the collapsed unfolded state of the small cold shock protein CspTm under near-native conditions. This regime is physiologically most relevant but difficult to access experimentally, because the equilibrium signal in ensemble experiments is dominated by folded molecules. Here, we avoid this problem in two ways. One is the use of single-molecule Fö rster resonance energy transfer, which allows the separation of folded and unfolded subpopulations at equilibrium and provides information on long-range intramolecular distance distributions. From experiments with donor and acceptor chromophores placed at different positions within the chain, we find that the distance distributions in unfolded CspTm agree surprisingly well with a Gaussian chain not only at high concentrations of denaturant, where the polypeptide chain is expanded, but also at low denaturant concentrations, where the chain is collapsed. The second, complementary approach is synchrotron radiation circular dichroism spectroscopy of collapsed unfolded molecules transiently populated with a microfluidic device that enables rapid mixing. The results indicate a ␤-structure content of the collapsed unfolded state of Ϸ20% compared with the folded protein. This suggests that collapse can induce secondary structure in an unfolded state without interfering with long-range distance distributions characteristic of a random coil, which were previously found only for highly expanded unfolded proteins.Gaussian chain ͉ microfluidic mixing ͉ protein folding ͉ random coil ͉ secondary structure W ith the discovery of small proteins that fold in the absence of populated intermediates (1), our quantitative understanding of the elementary properties of protein folding reactions has made significant advances, including the structural characterization of transition states for folding (2) and the prediction of folding rates from native structure (3-5). One of the most severe limitations for the further development of these approaches is our ignorance about the energetic or structural properties of unfolded † † states of proteins. Because of the structural heterogeneity and complexity of the ensembles of conformations populated by unfolded proteins, their experimental characterization has proven extremely difficult. Traditional methods, such as small-angle scattering techniques (6), provide only global physical properties, e.g., the radius of gyration. In some cases, more detailed structural information can be obtained from NMR (7-10), but these studies usually provide information about the denatured state only under nonnative conditions, typically in the presence of large concentrations of denaturant, or through severe destabilization of the native state induced by covalent modification or mutations. The most interesting and physiologically relevant situation, however, is that of an unfolded sta...