Kinetics are probe-dependent during downhill folding of an engineered λ _6–85 protein

Abstract

The Y22W͞Q33Y͞G46,48A mutant of the protein 6 -85 folds in a few microseconds at room temperature. We find that its folding kinetics are probe-dependent under a strong bias toward the native state, a new signature for downhill folding. The IR-and fluorescence-detected relaxation time scales converge when the native bias is removed by raising the temperature, recovering activated two-state folding. Langevin dynamics simulations on one-and 2D free energy surfaces tunable from two-state to downhill folding reproduce the difference between the IR and fluorescence experiments, as well as the temperature and viscosity trends. In addition, the 2D surface reproduces the stretched exponential dynamics that we fit to the glucose solution experimental data at short times. Nonexponential dynamics at <10 s is a signature either for local free energy minima along the reaction coordinate (''longitudinal roughness''), or for folding on a higherdimensional free energy surface (''transverse roughness'').fluorescence ͉ infrared ͉ helix bundle ͉ amide band ͉ landscape roughness T he possibility of downhill (type 0) folding is one of the central but least expected predictions of the energy landscape theory. As discussed by Bryngelson et al. (1), folding could proceed downhill in free energy when there is a sufficiently strong bias toward the native state. When the bias is reduced by heat denaturation, activated folding over a barrier (the type 1 scenario) is recovered (1). This switch corresponds to a transition from potentially nonexponential diffusion on a rough free energy surface to activated kinetics with a single rate coefficient, k a . Type 1 folding seems to be the norm among natural proteins, but type 0 folding remains a possibility for proteins with an unusually strong native bias.Significant experimental progress has been made in the search for downhill folding kinetics (2). With the advent of fast laser initiation techniques (3), including laser T-jumps (4-6), the necessary nano-to microsecond time scales have become accessible. In 1999, Sabelko et al. (7) reported downhill formation of a phosphoglycerate kinase (PGK) folding intermediate. The nonexponential dynamics could be tuned toward a single exponential by cold or heat denaturing the protein, decreasing the bias toward the native state. The C-terminal domain of PGK was later shown to be responsible for this behavior (8). Kinetic evidence indicating downhill folding to the native state has also been reported. Activated kinetics of the engineered 6-85 protein were preceded by a fast (k m Ϸ 1 s Ϫ1 ) ''molecular phase'' (9). This new phase was attributed to a substantial population en route between the native and denatured states and accounted for nearly the entire signal in stabilizing glucose solution (10). The experimental downhill folding ''speed limit'' is in agreement with the preexponential factor calculated by Portman et al. (11). Estimates based on diffusion of denatured proteins (12, 13), on generic folding models (14), and on molecular dynamics studies of th...

Cooperative folding near the downhill limit determined with amino acid resolution by hydrogen exchange

“…S2). Rapid CD and fluorescence measurements before aggregation suggest thermal denaturation is adequately described as proceeding through a single transition, consistent with Ma and Gruebele's findings (20). Whereas two-state, barrier-limited folding behavior near the thermal or urea-induced melting transition is possible even for downhill folders (24), the major issue under investigation is whether the cooperativity persists even under highly stabilizing conditions, which we will determine using HX.…”

“…Key signatures of downhill folding are the presence of kinetics that are on the same timescale as the chain dynamics, suggestive of a free energy barrier with nearzero activation energy, and a fast "molecular phase" reflecting the rapid redistribution of species near the top of the barrier after the T-jump that is more pronounced for the faster-folding variants (8). Additional signatures include probe-dependent kinetics (20) and aggregation tendencies (6). Lapidus and coworkers proposed that the λ D14A variant folds in a downhill manner based on a combination of microfluidic mixing measurements and probe-dependent melting temperatures (10).…”

Exploiting the downhill folding regime via experiment

“…Likewise, the observed relaxation time could become highly dependent on the structural probe employed, or on the diffusive path. This could be the regime observed by Gruebele and co-workers on ultrafast folding variants of repressor (Ma and Gruebele, 2005;Yang and Gruebele, 2004), and Tokmakoff and co-workers in ubiquitin (Chung and Tokmakoff, 2008).…”

Dynamics of one-state downhill protein folding

“…However, the probe-dependent equilibrium of one-state folding should correspond exactly with probe dependent amplitudes in relaxation experiments. Recently, it has been proposed that relaxation times may also exhibit probe dependence in rough downhill free energy surfaces (25). Coarsegrained off-lattice simulations have shown flat or even inverted chevron plots (i.e., plots of folding relaxation rate versus chemical denaturant concentration) for one-state folding (19).…”