Folding Kinetics of a Fluorescent Variant of Monomeric λ Repressor

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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...

Section: Methodsmentioning

confidence: 99%

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

Gruebele

2005

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“…T he nature of the energy landscape when folding occurs on the microsecond timescale continues to be investigated using both experimental and computational approaches. The 80-residue subdomain of the λ-repressor transcription factor, λ (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11), is an appealing system as it contains five α-helices arranged in a nonsymmetric pattern, folds in microseconds, and is nearly a downhill folder (12), a condition where a continuous series of folding events may be characterized. These characteristics along with a folding time that nears the upper limit for current all-atom simulations (13)(14)(15)(16)(17)(18)(19) make this protein appropriate for detailed comparisons between experiment and simulations.…”

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confidence: 99%

“…Oas and coworkers found that the wild type and a fasterfolding mutant with two helix-promoting substitutions (λ AA with G46A/G48A, Fig. S1A) fold cooperatively (1)(2)(3)(4). The transition state ensemble (TSE) characterized using ϕ analysis minimally contains α1 and α4 (3).…”

mentioning

confidence: 99%

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

Baxa

Gagnon

et al. 2016

The relationship between folding cooperativity and downhill, or barrier-free, folding of proteins under highly stabilizing conditions remains an unresolved topic, especially for proteins such as λ-repressor that fold on the microsecond timescale. Under aqueous conditions where downhill folding is most likely to occur, we measure the stability of multiple H bonds, using hydrogen exchange (HX) in a λ YA variant that is suggested to be an incipient downhill folder having an extrapolated folding rate constant of 2 × 10 5 s −1 and a stability of 7.4 kcal·mol −1 at 298 K. At least one H bond on each of the three largest helices (α1, α3, and α4) breaks during a common unfolding event that reflects global denaturation. The use of HX enables us to both examine folding under highly stabilizing, native-like conditions and probe the pretransition state region for stable species without the need to initiate the folding reaction. The equivalence of the stability determined at zero and high denaturant indicates that any residual denatured state structure minimally affects the stability even under native conditions. Using our ψ analysis method along with mutational ϕ analysis, we find that the three aforementioned helices are all present in the folding transition state. Hence, the free energy surface has a sufficiently high barrier separating the denatured and native states that folding appears cooperative even under extremely stable and fast folding conditions. T he nature of the energy landscape when folding occurs on the microsecond timescale continues to be investigated using both experimental and computational approaches. The 80-residue subdomain of the λ-repressor transcription factor, λ 6-85 (1-11), is an appealing system as it contains five α-helices arranged in a nonsymmetric pattern, folds in microseconds, and is nearly a downhill folder (12), a condition where a continuous series of folding events may be characterized. These characteristics along with a folding time that nears the upper limit for current all-atom simulations (13-19) make this protein appropriate for detailed comparisons between experiment and simulations.The energy landscape of λ 6-85 is significantly influenced by its sequence. Oas and coworkers found that the wild type and a fasterfolding mutant with two helix-promoting substitutions (λ AA with G46A/G48A, Fig. S1A) fold cooperatively (1-4). The transition state ensemble (TSE) characterized using ϕ analysis minimally contains α1 and α4 (3). Kinetic H/D isotope effect measurements in the Sosnick laboratory found that both λ 6-85 and λ AA fold cooperatively with ∼70% of the H bonds being formed in the TSE, matching the degree of surface buried in the TSE [beta Tanford value (β Tanford )] (5).Using nanosecond T-jump and other methods, Gruebele and coworkers proposed that faster-folding and stabilized variants, such as λ YA (λ AA + Q33Y) and λ D14A (λ YA + D14A), fold in an incipient or fully downhill manner, especially under highly stable conditions at lower temperatures (6-9). Key signatures of downhi...

“…We chose λ*YA, the Y22W/Q33Y/G46,48A mutant of λ-repressor fragment 6-85, as our model protein (11,12). Tryptophan W22 provides a fluorescent probe (11).…”

mentioning

confidence: 99%

“…We chose λ*YA, the Y22W/Q33Y/G46,48A mutant of λ-repressor fragment 6-85, as our model protein (11,12). Tryptophan W22 provides a fluorescent probe (11). Based on the crystal structure, tyrosine Y33 enhances the fluorescence lifetime difference between folded and unfolded states by contact quenching W22 when the protein is folded (13).…”

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confidence: 99%

Misplaced helix slows down ultrafast pressure-jump protein folding

Prigozhin¹,

Liu²,

Wirth³

et al. 2013

Using a newly developed microsecond pressure-jump apparatus, we monitor the refolding kinetics of the helix-stabilized five-helix bundle protein λ*YA, the Y22W/Q33Y/G46,48A mutant of λ-repressor fragment 6-85, from 3 μs to 5 ms after a 1,200-bar P-drop. In addition to a microsecond phase, we observe a slower 1.4-ms phase during refolding to the native state. Unlike temperature denaturation, pressure denaturation produces a highly reversible helix-coilrich state. This difference highlights the importance of the denatured initial condition in folding experiments and leads us to assign a compact nonnative helical trap as the reason for slower P-jumpinduced refolding. To complement the experiments, we performed over 50 μs of all-atom molecular dynamics P-drop refolding simulations with four different force fields. Two of the force fields yield compact nonnative states with misplaced α-helix content within a few microseconds of the P-drop. Our overall conclusion from experiment and simulation is that the pressure-denatured state of λ*YA contains mainly residual helix and little β-sheet; following a fast P-drop, at least some λ*YA forms misplaced helical structure within microseconds. We hypothesize that nonnative helix at helix-turn interfaces traps the protein in compact nonnative conformations. These traps delay the folding of at least some of the population for 1.4 ms en route to the native state. Based on molecular dynamics, we predict specific mutations at the helix-turn interfaces that should speed up refolding from the pressure-denatured state, if this hypothesis is correct.downhill folding | fluorescence lifetime | molecular dynamics simulation | thermal denaturation | lambda repressor T emperature and pressure are excellent perturbations when comparing experimental folding kinetics with molecular dynamics (MD) simulations (1). Fast temperature-jumps (T-jumps) and pressure-jumps (P-jumps) are relatively easy to simulate by MD. Typical temperature changes required to cross the protein folding transition are 5-20 K, easily implemented with laser T-jumps (2). Typical pressure changes required to cross the folding transition are 1-5 kbar, previously achieved only with millisecond time resolution (piezo methods are limited to ΔP < 100 bar) (3, 4). We recently reported a P-jump instrument capable of >1-kbar P-drops with ∼1-μs dead time (5).Folded proteins have a larger partial molar volume than pressure-denatured proteins (by about 10 1 -10 2 mL/mol) (6). The fractal dimension of their folded state is less than 3 because voids occur whenever a connected chain made from a finite amino acid alphabet is packed into a compact structure (7,8). Such imperfections in packing, which disappear when small water molecules solvate the polypeptide chain, lead to protein unfolding under pressure (9). Pressure unfolding is a slow process because the positive activation volume is unfavorable at high pressure (10).Here, we study the much faster process of protein refolding at 1 bar and room temperature, starting from the pressure-d...