Even with nonnative interactions, the updated folding transition states of the homologs Proteins G &amp; L are extensive and similar

Baxa, Michael C.; Yu, Wookyung; Adhikari, Aashish N.; Ge, Liang; Xia, Zhen; Zhou, Ruhong; Freed, Karl F.; Sosnick, Tobin R.

doi:10.1073/pnas.1503613112

Cited by 22 publications

(38 citation statements)

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“…The present study produces a picture of an extensive TSE, as observed for six other proteins studied using ψ (36,37,41,(48)(49)(50). Their TSEs adopt a high fraction of the native topology, defined using the relative contact order (RCO) parameter, RCO TSE ∼ 0.7·RCO N .…”

Section: Discussionmentioning

confidence: 69%

“…4 and Table S3). The addition of zinc or nickel ions, which coordinate the pair of histidines, alters the protein's stability and activation free energy for folding (ΔΔG eq and ΔΔG f , respectively) due to differences in binding constants K DSE , K N , and K TSE (35)(36)(37). The ion-induced changes in ΔΔG eq and ΔΔG f are used to define the ψ value, a parameter analogous to the mutational ϕ value, with ψ being the instantaneous change in ΔΔG f relative to ΔΔG eq ,…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

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

Baxa

Gagnon

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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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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Section: Discussionmentioning

confidence: 69%

Section: Resultsmentioning

confidence: 99%

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

Baxa

Gagnon

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Previous work has already shown that a single mutation may greatly affect the structure, stability or function of a protein [ 3 , 4 ], but some proteins significantly differ in sequence are found to share very similar structures and functions [ 1 , 2 ]. To unravel this mystery, folding of homologous proteins has been compared [ 5 , 6 ] with peripheral subunit binding domains [ 7 , 8 ], homologs protein G & L [ 9 ], spectrin domain R15, R16 and R17 [ 1 ] and RNase family [ 10 ]. It is noticed that some homologues share very similar folding mechanism [ 5 , 9 ], while some others significantly differ thermodynamically and kinetically [ 11 – 14 ].…”

Section: Introductionmentioning

confidence: 99%

Kinetic and thermodynamic studies reveal chemokine homologues CC11 and CC24 with an almost identical tertiary structure have different folding pathways

Jiang

Chen

et al. 2017

BMC Biophys

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BackgroundProteins with low sequence identity but almost identical tertiary structure and function have been valuable to uncover the relationship between sequence, tertiary structure, folding mechanism and functions. Two homologous chemokines, CCL11 and CCL24, with low sequence identity but similar tertiary structure and function, provide an excellent model system for respective studies.ResultsThe kinetics and thermodynamics of the two homologous chemokines were systematically characterized. Despite their similar tertiary structures, CCL11 and CCL24 show different thermodynamic stability in guanidine hydrochloride titration, with D50% = 2.20 M and 4.96 M, respectively. The kinetics curves clearly show two phases in the folding/unfolding processes of both CCL11 and CCL24, which suggests the existence of an intermediate state in their folding/unfolding processes. The folding pathway of both CCL11 and CCL24 could be well described using a folding model with an on-pathway folding intermediate. However, the folding kinetics and stability of the intermediate state of CCL11 and CCL24 are obviously different.ConclusionOur results suggest homologous proteins with low sequence identity can display almost identical tertiary structure, but very different folding mechanisms, which applies to homologues in the chemokine protein family, extending the general applicability of the above observation.Electronic supplementary materialThe online version of this article (10.1186/s13628-017-0039-4) contains supplementary material, which is available to authorized users.

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“…This leads to an off-pathway kinetic trap. Although previous work has also identified nonnative states involving these hairpins docked in an anti-parallel orientation [38], our work provides a quantitative estimate of the effect of these nonnative contacts on folding kinetics. Namely, our finding that a significant fraction of the population adopts long-lived states in which the hairpins misalign in an antiparallel fashion ( Fig.…”

Section: Use Of Dbfold To Generate Experimentally-testable Predictionsmentioning

confidence: 85%

“…As a proof of principle, we now apply our method on Streptocaccal Protein G, a model protein whose folding has been extensively studied using both computational and experimental methods [20,[31][32][33][34][35][36][37][38]. We begin by running equilibrium simulations with replica exchange and umbrella sampling using native contacts as the reaction coordinate along which we bias [39] (See Materials and methods).…”

Section: Computing Equilibrium Folding Properties For Protein Gmentioning

confidence: 99%

DBFOLD: An efficient algorithm for computing folding pathways of complex proteins

Bitran

Jacobs²,

Shakhnovich

2020

Preprint

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Atomistic simulations can provide valuable, experimentally-verifiable insights into protein folding mechanisms, but existing ab initio simulation methods are restricted to only the smallest proteins due to severe computational speed limits. The folding of larger proteins has been studied using native-centric potential functions, but such models omit the potentially crucial role of non-native interactions.Here, we present an algorithm, entitled DBFOLD, which can predict folding pathways for a wide range of proteins while accounting for the effects of non-native contacts. In addition, DBFOLD can predict the relative rates of different transitions within a protein’s folding pathway. To accomplish this, rather than directly simulating folding, our method combines equilibrium Monte-Carlo simulations, which deploy enhanced sampling, with unfolding simulations at high temperatures. We show that under certain conditions, trajectories from these two types of simulations can be jointly analyzed to compute unknown folding rates from detailed balance. This requires inferring free energies from the equilibrium simulations, and extrapolating transition rates from the unfolding simulations to lower, physiologically-reasonable temperatures at which the native state is marginally stable. As a proof of principle, we show that our method can accurately predict folding pathways and Monte-Carlo rates for the well-characterized Streptococcal protein G. We then show that our method significantly reduces the amount of computation time required to compute the folding pathways of large, misfolding-prone proteins that lie beyond the reach of existing direct simulation methods. Our algorithm, which is available online, can generate detailed atomistic models of protein folding mechanisms while shedding light on the role of non-native intermediates which may crucially affect organismal fitness and are frequently implicated in disease.Author summaryMany proteins must adopt a specific structure in order to function. Computational simulations have been used to shed light on the mechanisms of protein folding, but unfortunately, realistic simulations can typically only be run for small proteins, due to severe limits in computational speed. Here, we present a method to solve this problem, whereby instead of directly simulating folding from an unfolded state, we run simulations that allow for computation of equilibrium folding free energies, alongside high temperature simulations to compute unfolding rates. From these quantities, folding rates can be computed using detailed balance. Importantly, our method can account for the effects of nonnative contacts which transiently form during folding and must be broken prior to adoption of the native state. Such contacts, which are often excluded from simple models of folding, may crucially affect real protein folding pathways and are often observed in folding intermediates implicated in disease.

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Even with nonnative interactions, the updated folding transition states of the homologs Proteins G & L are extensive and similar

Cited by 22 publications

References 68 publications

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

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

Kinetic and thermodynamic studies reveal chemokine homologues CC11 and CC24 with an almost identical tertiary structure have different folding pathways

DBFOLD: An efficient algorithm for computing folding pathways of complex proteins

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