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There has been a long-standing controversy regarding the effect of chemical denaturants on the dimensions of unfolded and intrinsically disordered proteins: A wide range of experimental techniques suggest that polypeptide chains expand with increasing denaturant concentration, but several studies using small-angle X-ray scattering (SAXS) have reported no: such increase of the radius of gyration (R-g). This inconsistency challenges our current understanding of the mechanism of chemical denaturants, which are widely employed to investigate protein folding and stability. Here, we use a combination Of single-molecule Forster resonance energy transfer (FRET), SAXS, dynamic light scattering (DLS), and two-focus fluorescence correlation spectroscopy (2f-FCS) to characterize the denaturant dependence of the unfolded state of the spectrin domain R17 and the intrinsically disordered protein ACTR in two different denaturants. Standard analysis of the primary data clearly indicates an expansion of the unfolded state with increasing denaturant concentration irrespective of the protein, denaturant, or experimental method used. This is the first case in which SAXS and FRET have yielded even qualitatively consistent results regarding expansion in denaturant when applied to the same proteins. To more directly illustrate this self-consistency, we used both SAXS and FRET data in a Bayesian procedure to refine structural ensembles representative of the observed unfolded state. This analysis demonstrates that both of these experimental probes are compatible with a common ensemble of protein configurations for each denaturant concentration. Furthermore, the resulting ensembles reproduce the trend of increasing hydrodynamic radius, with denaturant concentration obtained by 2f-FCS,and DLS. We were thus able to reconcile the results from all four experimental techniques quantitatively, to obtain a comprehensive structural picture of denaturant;induced unfolded state expansion, and to identify the Most likely sources of earlier discrepancies.
Quenching of the triplet state of tryptophan by cysteine has provided a new tool for measuring the rate of forming a specific intramolecular contact in disordered polypeptides. Here, we use this technique to investigate contact formation in the denatured state of CspTm, a small cold-shock protein from Thermotoga maritima, engineered to contain a single tryptophan residue (W29) and a single cysteine residue at the C terminus (C67). At all concentrations of denaturant, the decay rate of the W29 triplet of the unfolded protein is more than tenfold faster than the rate observed for the native protein (not, vert, similar104 s;1). Experiments on the unfolded protein without the added C- terminal cysteine residue show that this faster rate results entirely from contact quenching by C67. The quenching rate in the unfolded state by C67 increases at concentrations of denaturant that favor folding, indicating a compaction of the unfolded protein as observed previously in single-molecule Foerster resonance energy transfer (FRET) experiments.
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 T-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.
We have developed a microfluidic mixer optimized for rapid measurements of protein folding kinetics using synchrotron radiation circular dichroism (SRCD) spectroscopy. The combination of fabrication in fused silica and synchrotron radiation allows measurements at wavelengths below 220 nm, the typical limit of commercial instrumentation. At these wavelengths, the discrimination between the different types of protein secondary structure increases sharply. The device was optimized for rapid mixing at moderate sample consumption by employing a serpentine channel design, resulting in a dead time of less than 200 ;s. Here, we discuss the design and fabrication of the mixer and quantify the mixing efficiency using wide-field and confocal epi-fluorescence microscopy. We demonstrate the performance of the device in SRCD measurements of the folding kinetics of cytochrome c, a small, fast-folding protein. Our results show that the combination of SRCD with microfluidic mixing opens new possibilities for investigating rapid conformational changes in biological macromolecules that have previously been inaccessible.
In order to investigate the behavior of single molecules under conditions far from equilibrium, we have coupled a microfabricated laminar-flow mixer to a confocal optical system. This combination enables time-resolved measurement of Foerster resonance energy transfer after an abrupt change in solution conditions. Observations of a small protein show the evolution of the intramolecular distance distribution as folding progresses. This technique can expose subpopulations, such as unfolded protein under conditions favoring the native structure, that would be obscured in equilibrium experiments.
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 singlemolecule FRET, this question can be addressed even under nearnative 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 temperaturedependent interactions within the unfolded chain. The observation of a collapse of a similar extent in the extremely hydrophilic, intrinsically disordered protein prothymosin 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.