A single-sweep, nanosecond time resolution laser temperature-jump apparatus

144

224

A kinetic and thermodynamic survey of 35 WW domain sequences is used in combination with a model to discern the energetic requirements for the transition from two-state folding to downhill folding. The sequences used exhibit a 600-fold range of folding rates at the temperature of maximum folding rate. Very stable proteins can achieve complete downhill folding when the temperature is lowered sufficiently below the melting temperature, and then at even lower temperatures they become two-state folders again because of cold denaturation. Less stable proteins never achieve a sufficient bias to fold downhill because of the onset of cold denaturation. The model, considering both heat and cold denaturation, reveals that to achieve incipient downhill folding (barrier <3 RT) or downhill folding (no barrier), the WW domain average melting temperatures have to be >50°C for incipient downhill folding and >90°C for downhill folding.activated rate ͉ alkene peptide isosteres ͉ molecular rate ͉ speed limit ͉ stability E nergy landscape theory predicts that the entropic and enthalpic contributions to the free energy of a protein may be able to compensate for one another to the point where no significant (Ͼ3 RT) barrier appears along the folding reaction coordinate (1). Such folding is now referred to as ''type 0'' or ''downhill'' folding (1, 2). The possibility of downhill folding has been supported by a number of experiments (3-7). Kinetic measurements have focused on the transition from simple single exponential (two-state) to nonexponential (low barrier) back toward simpler (pure downhill) kinetics as the thermodynamic bias toward the native state is increased (3,6,8,9). Thermodynamic measurements with probe-dependent baselines and transition temperatures suggest that downhill folding may be possible even at the melting temperature of a protein (10-12). Recently, two engineered proteins with identical melting temperatures were compared by both kinetic and thermodynamic criteria, showing that one can be classified as a two-state folder, whereas the other can be classified as a downhill folder (13). Such results have been debated extensively in the literature (14-17).It has been suggested that the kinetics and thermodynamics of downhill folders must be very sensitive to sequence and environment because of the low barriers involved (18). Indeed, the fast folder lambda repressor has been shown to fold by either two-state, framework intermediate, or downhill mechanisms, depending on solvent conditions and sequence (6,9,13,19,20). Models for protein BBL, another fast folding protein, also indicate that it folds either in a two-state or downhill manner, depending on the exact sequence and solvent conditions (21,22).The diversity of observations suggests that criteria for downhill folding can be developed only by examining a large number of fast-folding proteins. Here, we take a survey approach to the experimental study of downhill folding. We examine a series of 35 engineered WW domains with variation in loops 1 and 2, where sequence...

“…The evolution of the fluorescence lifetime profile was extracted by analysis as described in ref. 38, to reveal deviations from two-state folding via nonexponential decays (Fig. 3).…”

Section: Methodsmentioning

confidence: 99%

An experimental survey of the transition between two-state and downhill protein folding scenarios

Fuller

et al. 2008

144

224

“…20 and 21. For the highest signal-to-noise ratio fluorescence transients, analysis (linear decomposition) was also used (22).…”

Section: Methodsmentioning

confidence: 99%

Kinetics are probe-dependent during downhill folding of an engineered λ_6–85protein

Gruebele

2005

101

140

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...

“…Artifact Tests. Our conditions were selected so tryptophan fluorescence was constant 10 ns-10 ms after the T-jump, eliminating T-jump and recooling artifacts (27). A small Ϸ10-s fluorescence-quenching phase during relaxation of pH 2 PGK (which is unfolded over the entire T range) was assigned to nonspecific contraction of the polypeptide chain (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…The nanosecond laser temperature jump (T-jump) apparatus used in our refolding experiments has been described previously (27,28). Degassed and filtered aqueous PGK and Ub*G solutions were jumped from ϷϪ8°C to temperatures between 0 and 20°C by a 1.54 m infrared laser pulse.…”

Section: Methodsmentioning

confidence: 99%

Observation of strange kinetics in protein folding

Sabelko

Ervin

Gruebele

1999

Self Cite

306

302

Highly nonexponential folding kinetics in aqueous solution have been observed during temperature jump-induced refolding of two proteins, yeast phosphoglycerate kinase and a ubiquitin mutant. The observations are most easily interpreted in terms of downhill folding, which posits a heterogeneous ensemble of structures en route to the folded state. The data are also reconciled with exponential kinetics measured under different experimental conditions and with titration experiments indicating cooperative folding.