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

Abstract

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...

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“…This is consistent with our prediction using the single set of long simulations. Additionally, this is what has been seen with previous simulation reports, , and hydrogen exchange experiments . Interestingly, this is counter to the results from a Go̅ model, which has been previously used and describes helices 1–4 folding cooperatively .…”

“…Additionally, this is what has been seen with previous simulation reports, 57,58 and hydrogen exchange experiments. 59 Interestingly, this is counter to the results from a Gō model, which has been previously used and describes helices 1-4 folding cooperatively. 60 This difference suggests that FAST-contacts is not simply an expensive Gō model.…”

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