Benjamin Schuler

Probing the free-energy surface for protein folding with single-molecule fluorescence spectroscopy

Protein folding is inherently a heterogeneous process because of the very large number of microscopic pathways that connect the myriad unfolded conformations to the unique conformation of the native structure. In a first step towards the long-range goal of describing the distribution of pathways experimentally, Förster resonance energy transfer (FRET) has been measured on single, freely diffusing molecules. Here we use this method to determine properties of the free-energy surface for folding that have not been obtained from ensemble experiments. We show that single-molecule FRET measurements of a small cold-shock protein expose equilibrium collapse of the unfolded polypeptide and allow us to calculate limits on the polypeptide reconfiguration time. From these results, limits on the height of the free-energy barrier to folding are obtained that are consistent with a simple statistical mechanical model, but not with the barriers derived from simulations using molecular dynamics. Unlike the activation energy, the free-energy barrier includes the activation entropy and thus has been elusive to experimental determination for any kinetic process in solution.

Protein folding studied by single-molecule FRET.

A complete understanding of a protein-folding mechanism requires description of the distribution of microscopic pathways that connect the folded and unfolded states. This distribution can, in principle, be described by computer simulations and theoretical models of protein folding, but is hidden in conventional experiments on large ensembles of molecules because only average properties are measured. A long-term goal of single-molecule fluorescence studies is to time-resolve the structural events as individual molecules make transitions between folded and unfolded states. Although such studies are still in their infancy, the work till now shows great promise and has already produced novel and important information on current issues in protein folding that has been impossible or difficult to obtain from ensemble measurements.

Ultrafast dynamics of protein collapse from single-molecule photon statistics

We use the statistics of photon emission from single molecules to probe the ultrafast dynamics of an unfolded protein via Förster resonance energy transfer. Global reconfiguration of the chain occurs on a time scale of ≈50 ns and slows down concomitant with chain collapse under folding conditions. These diffusive dynamics provide a missing link between the phenomenological chemical kinetics commonly used in protein folding and a physical description in terms of quantitative free energy surfaces. The experiments demonstrate the potential of single-molecule methods in accessing the biologically important nanosecond time scales even in heterogeneous populations.

From the Cover: Charge interactions can dominate the dimensions of intrinsically disordered proteins

Many eukaryotic proteins are disordered under physiological conditions, and fold into ordered structures only on binding to their cellular targets. Such intrinsically disordered proteins (IDPs) often contain a large fraction of charged amino acids. Here, we use single-molecule Förster resonance energy transfer to investigate the influence of charged residues on the dimensions of unfolded and intrinsically disordered proteins. We find that, in contrast to the compact unfolded conformations that have been observed for many proteins at low denaturant concentration, IDPs can exhibit a prominent expansion at low ionic strength that correlates with their net charge. Charge-balanced polypeptides, however, can exhibit an additional collapse at low ionic strength, as predicted by polyampholyte theory from the attraction between opposite charges in the chain. The pronounced effect of charges on the dimensions of unfolded proteins has important implications for the cellular functions of IDPs.

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

We have used the combination of single-molecule Förster 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 Förster 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 β-structure content of the collapsed unfolded state of ≈20% 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.

Quantifying internal friction in unfolded and intrinsically disordered proteins with single-molecule spectroscopy

Internal friction, which reflects the “roughness” of the energy landscape, plays an important role for proteins by modulating the dynamics of their folding and other conformational changes. However, the experimental quantification of internal friction and its contribution to folding dynamics has remained challenging. Here we use the combination of single-molecule Förster resonance energy transfer, nanosecond fluorescence correlation spectroscopy, and microfluidic mixing to determine the reconfiguration times of unfolded proteins and investigate the mechanisms of internal friction contributing to their dynamics. Using concepts from polymer dynamics, we determine internal friction with three complementary, largely independent, and consistent approaches as an additive contribution to the reconfiguration time of the unfolded state. We find that the magnitude of internal friction correlates with the compactness of the unfolded protein: its contribution dominates the reconfiguration time of approximately 100 ns of the compact unfolded state of a small cold shock protein under native conditions, but decreases for more expanded chains, and approaches zero both at high denaturant concentrations and in intrinsically disordered proteins that are expanded due to intramolecular charge repulsion. Our results suggest that internal friction in the unfolded state will be particularly relevant for the kinetics of proteins that fold in the microsecond range or faster. The low internal friction in expanded intrinsically disordered proteins may have implications for the dynamics of their interactions with cellular binding partners.

Single-molecule spectroscopy of protein folding dynamics--expanding scope and timescales.

Single-molecule spectroscopy has developed into an important method for probing protein structure and dynamics, especially in structurally heterogeneous systems. A broad range of questions in the diversifying field of protein folding have been addressed with single-molecule Förster resonance energy transfer (FRET) and photo-induced electron transfer (PET). Building on more than a decade of rapid method development, these techniques can now be used to investigate a wide span of timescales, an aspect that we focus on in this review. Important current topics range from the structure and dynamics of unfolded and intrinsically disordered proteins, including the coupling of folding and binding, to transition path times, the folding and misfolding of larger proteins, and their interactions with molecular chaperones.

Effect of flexibility and cis residues in single-molecule FRET studies of polyproline

Polyproline has recently been used as a spacer between donor and acceptor chromophores to help establish the accuracy of distances determined from single-molecule Förster resonance energy transfer (FRET) measurements. This work showed that the FRET efficiency in water is higher than expected for a rigid spacer and was attributed to the flexibility of the polypeptide. Here, we investigate this issue further, using a combination of single-molecule fluorescence intensity and lifetime measurements, NMR, theory, and molecular dynamics simulations of polyproline-20 that include the dyes and their linkers to the polypeptide. NMR shows that in water ≈30% of the molecules contain internal cis prolines, whereas none are detectable in trifluoroethanol. Simulations suggest that the all-trans form of polyproline is relatively stiff, with persistence lengths of 9–13 nm using different established force fields, and that the kinks arising from internal cis prolines are primarily responsible for the higher mean FRET efficiency in water. We show that the observed efficiency histograms and distributions of donor fluorescence lifetimes are explained by the presence of multiple species with efficiencies consistent with the simulations and populations determined by NMR. In calculating FRET efficiencies from the simulation, we find that the fluctuations of the chromophores, attached to long flexible linkers, also play an important role. A similar simulation approach suggests that the flexibility of the chromophore linkers is largely responsible for the previously unexplained high value of R0 required to fit the data in the classic study of Stryer and Haugland.

Single-molecule spectroscopy of the temperature-induced collapse of unfolded proteins.

We used single-molecule FRET in combination with other biophysical methods and molecular simulations to investigate the effect of temperature on the dimensions of unfolded proteins. With single-molecule FRET, this question can be addressed even under near-native conditions, where most molecules are folded, allowing us to probe a wide range of denaturant concentrations and temperatures. We find a compaction of the unfolded state of a small cold shock protein with increasing temperature in both the presence and the absence of denaturant, with good agreement between the results from single-molecule FRET and dynamic light scattering. Although dissociation of denaturant from the polypeptide chain with increasing temperature accounts for part of the compaction, the results indicate an important role for additional temperature-dependent interactions within the unfolded chain. The observation of a collapse of a similar extent in the extremely hydrophilic, intrinsically disordered protein prothymosin α suggests that the hydrophobic effect is not the sole source of the underlying interactions. Circular dichroism spectroscopy and replica exchange molecular dynamics simulations in explicit water show changes in secondary structure content with increasing temperature and suggest a contribution of intramolecular hydrogen bonding to unfolded state collapse.

Polymer scaling laws of unfolded and intrinsically disordered proteins quantified with single-molecule spectroscopy

The dimensions of unfolded and intrinsically disordered proteins are highly dependent on their amino acid composition and solution conditions, especially salt and denaturant concentration. However, the quantitative implications of this behavior have remained unclear, largely because the effective theta-state, the central reference point for the underlying polymer collapse transition, has eluded experimental determination. Here, we used single-molecule fluorescence spectroscopy and two-focus correlation spectroscopy to determine the theta points for six different proteins. While the scaling exponents of all proteins converge to 0.62 ± 0.03 at high denaturant concentrations, as expected for a polymer in good solvent, the scaling regime in water strongly depends on sequence composition. The resulting average scaling exponent of 0.46 ± 0.05 for the four foldable protein sequences in our study suggests that the aqueous cellular milieu is close to effective theta conditions for unfolded proteins. In contrast, two intrinsically disordered proteins do not reach the Θ-point under any of our solvent conditions, which may reflect the optimization of their expanded state for the interactions with cellular partners. Sequence analyses based on our results imply that foldable sequences with more compact unfolded states are a more recent result of protein evolution.