Biomolecules are highly pressure-sensitive, but their dynamics upon return to ambient pressure are often too fast to observe with existing approaches. We describe a sample-efficient method capable of large and very fast pressure drops (<1 nanomole, >2,500 atmospheres and <0.7 microseconds). We validated the method by fluorescence-detected refolding of a genetically engineered lambda repressor mutant from its pressure-denatured state. We resolved barrierless structure formation upon return to ambient pressure; we observed a 2.1 +/- 0.7 microsecond refolding time, which is very close to the 'speed limit' for proteins and much faster than the corresponding temperature-jump refolding of the same protein. The ability to experimentally perform a large and very fast pressure drop opens up a new region of the biomolecular energy landscape for atomic-level simulation.

The l à 6À85 pseudo-wild type of lambda repressor fragment is a fast two-state folder (k f % 35 msec À1 at 58°C). Previously, highly stable l à 6À85 mutants with k f > 30 msec À1 have been engineered to fold nearly or fully downhill. Stabilization of the native state by solvent tuning might also tune l à 6À85 away from two-state folding. We test this prediction by examining the folding thermodynamics and kinetics of l à 6À85 in a stabilizing solvent, 45% by weight aqueous ethylene glycol at -28°C. Detection of kinetics by circular dichroism at 222 nm (sensitive to helix content) and small angle X-ray scattering (measuring the radius of gyration) shows that refolding from guanidine hydrochloride denatured conditions exhibits very different time scales for collapse and secondary structure formation: the two processes become decoupled. Collapse remains a low-barrier activated process, while the fastest of several secondary structure formation time scales approaches the downhill folding limit. Two-state folding of l à 6À85 is not a robust process.

We present a new method for computing interaction potentials of solvated proteins directly from small-angle x-ray scattering data. An ensemble of proteins is modeled by Monte Carlo or molecular dynamics simulation. The global x-ray scattering of the whole model ensemble is then computed at each snapshot of the simulation, and averaged to obtain the x-ray scattering intensity. Finally, the interaction potential parameters are adjusted by an optimization algorithm, and the procedure is iterated until the best agreement between simulation and experiment is obtained. This new approach obviates the need for approximations that must be made in simplified analytical models. We apply the method to lambda repressor fragment 6-85 and fyn-SH3. With the increased availability of fast computer clusters, Monte Carlo and molecular dynamics analysis using residue-level or even atomistic potentials may soon become feasible.

The mutant Tyr22Trp/Glu33Tyr/Gly46Ala/Gly48Ala of lambda repressor fragment lambda(6-85) was previously assigned as an incipient downhill folder. We slow down its folding in a cryogenic water-ethylene-glycol solvent (-18 to -28 degrees C). The refolding kinetics are probed by small-angle x-ray scattering, circular dichroism, and fluorescence to measure the radius of gyration, the average secondary structure content, and the native packing around the single tryptophan residue. The main resolved kinetic phase of the mutant is probe independent and faster than the main phase observed for the pseudo-wild-type. Excess helical structure formed early on by the mutant may reduce the formation of turns and prevent the formation of compact misfolded states, speeding up the overall folding process. Extrapolation of our main cryogenic folding phase and previous T-jump measurements to 37 degrees C yields nearly the same refolding rate as extrapolated by Oas and co-workers from NMR line-shape data. Taken together, all the data consistently indicate a folding speed limit of approximately 4.5 micros for this fast folder.

We present fluorescence-detected measurements of the temperature-jump relaxation kinetics of the designed three-helix bundle protein ␣ 3 D taken under solvent conditions identical to previous infrared-detected kinetics. The fluorescence-detected rate is similar to the IR-detected rate only at the lowest temperature where we could measure it ͑326 K͒. The fluorescence-detected rate decreases by a factor of 3 over the 326-344 K temperature range, whereas the IR-detected rate remains nearly constant over the same range. To investigate this probe dependence, we tested an extensive set of physically reasonable one-dimensional ͑1D͒ free energy surfaces by Langevin dynamics simulation. The simulations included coordinate-and temperature-dependent roughness, diffusion coefficients, and IR/fluorescence spectroscopic signatures. None of these can reproduce the IR and fluorescence data simultaneously, forcing us to the conclusion that a 1D free energy surface cannot accurately describe the folding of ␣ 3 D. This supports the hypothesis that ␣ 3 D has a multidimensional free energy surface conducive to downhill folding at 326 K, and that it is already an incipient downhill folder with probe-dependent kinetics near its melting point.