Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Fast pressure-jump all-atom simulations and experiments reveal site-specific protein dehydration-folding dynamics.

ABSTRACT: As theory and experiment have shown, protein dehydration is a major contributor to protein folding. Dehydration upon folding can be characterized directly by all-atom simulations of fast pressure drops, which create desolvated pockets inside the nascent hydrophobic core. Here, we study pressure-drop refolding of three ?-repressor fragment (?6-85) mutants computationally and experimentally. The three mutants report on tertiary structure formation via different fluorescent helix-helix contact pairs. All-atom simulations of pressure drops capture refolding and unfolding of all three mutants by a similar mechanism, thus validating the nonperturbative nature of the fluorescent contact probes. Analysis of simulated interprobe distances shows that the ?-helix 1-3 pair distance displays a slower characteristic time scale than the 1-2 or 3-2 pair distance. To see whether slow packing of ?-helices 1 and 3 is reflected in the rate-limiting folding step, fast pressure-drop relaxation experiments captured refolding on a millisecond time scale. These experiments reveal that refolding monitored by 1-3 contact formation indeed is much slower than when monitored by 1-2 or 3-2 contact formation. Unlike the case of the two-state folder [three-?-helix bundle (?3D)], whose drying and core formation proceed in concert, ?6-85 repeatedly dries and rewets different local tertiary contacts before finally forming a solvent-excluded core, explaining the non-two-state behavior observed during refolding in molecular dynamics simulations. This work demonstrates that proteins can explore desolvated pockets and dry globular states numerous times before reaching the native conformation.

SUBMITTER: Prigozhin MB

PROVIDER: S-EPMC6431218 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Comparing Fast Pressure Jump and Temperature Jump Protein Folding Experiments and Simulations.

Project description:The unimolecular folding reaction of small proteins is now amenable to a very direct mechanistic comparison between experiment and simulation. We present such a comparison of microsecond pressure and temperature jump refolding kinetics of the engineered WW domain FiP35, a model system for β-sheet folding. Both perturbations produce experimentally a faster and a slower kinetic phase, and the "slow" microsecond phase is activated. The fast phase shows differences between perturbation methods and is closer to the downhill limit by temperature jump, but closer to the transiently populated intermediate limit by pressure jump. These observations make more demands on simulations of the folding process than just a rough comparison of time scales. To complement experiments, we carried out several pressure jump and temperature jump all-atom molecular dynamics trajectories in explicit solvent, where FiP35 folded in five of the six simulations. We analyzed our pressure jump simulations by kinetic modeling and found that the pressure jump experiments and MD simulations are most consistent with a 4-state kinetic mechanism. Together, our experimental and computational data highlight FiP35's position at the boundary where activated intermediates and downhill folding meet, and we show that this model protein is an excellent candidate for further pressure jump molecular dynamics studies to compare experiment and modeling at the folding mechanism level.

| S-EPMC4794261 | biostudies-literature

Interrupted Pressure-Jump NMR Experiments Reveal Resonances of On-Pathway Protein Folding Intermediate.

Project description:Previous pressure-jump NMR experiments on a pressure-sensitized double mutant of ubiquitin showed evidence that its folding occurs via two parallel, comparably efficient pathways: a single barrier and a two-barrier pathway. An interrupted folding NMR experiment is introduced, where for a brief period the pressure is dropped to atmospheric conditions (1 bar), followed by a jump back to high pressure for signal detection. Conventional, forward sampling of the indirect dimension during the low-pressure period correlates the 15N or 13C' chemical shifts of the unfolded protein at 1 bar to the 1H frequencies of both the unfolded and folded proteins at high pressure. Remarkably, sampling the data of the same experiment in the reverse direction yields the frequencies of proteins present at the end of the low-pressure interval, which include unfolded, intermediate, and folded species. Although the folding intermediate 15N shifts differ strongly from natively folded protein, its 13C' chemical shifts, which are more sensitive probes for secondary structure, closely match those of the folded protein and indicate that the folding intermediate must have a structure that is quite similar to the native state.

| S-EPMC6698896 | biostudies-literature

Monitoring Hydrogen Exchange During Protein Folding by Fast Pressure Jump NMR Spectroscopy.

Project description:A method is introduced that permits direct observation of the rates at which backbone amide hydrogens become protected from solvent exchange after rapidly dropping the hydrostatic pressure inside the NMR sample cell from denaturing (2.5 kbar) to native (1 bar) conditions. The method is demonstrated for a pressure-sensitized ubiquitin variant that contains two Val to Ala mutations. Increased protection against hydrogen exchange with solvent is monitored as a function of time during the folding process. Results for 53 backbone amides show narrow clustering with protection occurring with a time constant of ca. 85 ms, but slower protection is observed around a reverse turn near the C-terminus of the protein. Remarkably, the native NMR spectrum returns with this slower time constant of ca. 150 ms, indicating that the almost fully folded protein retains molten globule characteristics with severe NMR line broadening until the final hydrogen bonds are formed. Prior to crossing the transition state barrier, hydrogen exchange protection factors are close to unity, but with slightly elevated values in the β1-β2 hairpin, previously shown to be already lowly populated in the urea-denatured state.

| S-EPMC5586491 | biostudies-literature

Single-molecule nonequilibrium periodic Mg2+-concentration jump experiments reveal details of the early folding pathways of a large RNA.

Project description:The evolution of RNA conformation with Mg(2+) concentration ([Mg(2+)]) is typically determined from equilibrium titration measurements or nonequilibrium single [Mg(2+)]-jump measurements. We study the folding of single RNA molecules in response to a series of periodic [Mg(2+)] jumps. The 260-residue catalytic domain of RNase P RNA from Bacillus stearothermophilus is immobilized in a microfluidic flow chamber, and the RNA conformational changes are probed by fluorescence resonance energy transfer (FRET). The kinetics of population redistribution after a [Mg(2+)] jump and the observed connectivity of FRET states reveal details of the folding pathway that complement and transcend information from equilibrium or single-jump measurements. FRET trajectories for jumps from [Mg(2+)] = 0.01 to 0.1 mM exhibit two-state behavior whereas jumps from 0.01 mM to 0.4 mM exhibit two-state unfolding but multistate folding behavior. RNA molecules in the low and high FRET states before the [Mg(2+)] increase are observed to undergo dynamics in two distinct regions of the free energy landscape separated by a high barrier. We describe the RNA structural changes involved in crossing this barrier as a "hidden" degree of freedom because the changes do not alter the detected FRET value but do alter the observed dynamics. The associated memory prevents the populations from achieving their equilibrium values at the end of the 5- to 10-sec [Mg(2+)] interval, thereby creating a nonequilibrium steady-state condition. The capability of interrogating nonequilibrium steady-state RNA conformations and the adjustable period of [Mg(2+)]-jump cycles makes it possible to probe regions of the free energy landscape that are infrequently sampled in equilibrium or single-jump measurements.

| S-EPMC2373323 | biostudies-literature

Misplaced helix slows down ultrafast pressure-jump protein folding.

Project description:Using a newly developed microsecond pressure-jump apparatus, we monitor the refolding kinetics of the helix-stabilized five-helix bundle protein ?*YA, the Y22W/Q33Y/G46,48A mutant of ?-repressor fragment 6-85, from 3 ?s 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-coil-rich 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-jump-induced refolding. To complement the experiments, we performed over 50 ?s 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 ?-helix content within a few microseconds of the P-drop. Our overall conclusion from experiment and simulation is that the pressure-denatured state of ?*YA contains mainly residual helix and little ?-sheet; following a fast P-drop, at least some ?*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.

| S-EPMC3657825 | biostudies-literature

Rapid folding of the prion protein captured by pressure-jump.

Project description:The conversion of the cellular form of the prion protein (PrP(C)) to an altered disease state, generally denoted as scrapie isoform (PrP(Sc)), appears to be a crucial molecular event in prion diseases. The details of this conformational transition are not fully understood, but it is perceived that they are associated with misfolding of PrP or its incapacity to maintain the native fold during its cell cycle. Here we present a tryptophan mutant of PrP (F198W), which has enhanced fluorescence sensitivity to unfolding/refolding transitions. Equilibrium folding was studied by circular dichroism and fluorescence. Pressure-jump experiments were successfully applied to reveal rapid submillisecond folding events of PrP at temperatures not accessed before.

| S-EPMC4509520 | biostudies-literature

Microsecond folding experiments and simulations: a match is made.

Project description:For the past two decades, protein folding experiments have been speeding up from the second or millisecond time scale to the microsecond time scale, and full-atom simulations have been extended from the nanosecond to the microsecond and even millisecond time scale. Where the two meet, it is now possible to compare results directly, allowing force fields to be validated and refined, and allowing experimental data to be interpreted in atomistic detail. In this perspective we compare recent experiments and simulations on the microsecond time scale, pointing out the progress that has been made in determining native structures from physics-based simulations, refining experiments and simulations to provide more quantitative underlying mechanisms, and tackling the problems of multiple reaction coordinates, downhill folding, and complex underlying structure of unfolded or misfolded states.

| S-EPMC3632410 | biostudies-literature

Fast machine-learning online optimization of ultra-cold-atom experiments.

Project description:We apply an online optimization process based on machine learning to the production of Bose-Einstein condensates (BEC). BEC is typically created with an exponential evaporation ramp that is optimal for ergodic dynamics with two-body s-wave interactions and no other loss rates, but likely sub-optimal for real experiments. Through repeated machine-controlled scientific experimentation and observations our 'learner' discovers an optimal evaporation ramp for BEC production. In contrast to previous work, our learner uses a Gaussian process to develop a statistical model of the relationship between the parameters it controls and the quality of the BEC produced. We demonstrate that the Gaussian process machine learner is able to discover a ramp that produces high quality BECs in 10 times fewer iterations than a previously used online optimization technique. Furthermore, we show the internal model developed can be used to determine which parameters are essential in BEC creation and which are unimportant, providing insight into the optimization process of the system.

| S-EPMC4867626 | biostudies-literature

Dual folding pathways of an alpha/beta protein from all-atom ab initio folding simulations.

Project description:Successful ab initio folding of proteins with both alpha-helix and beta-sheet requires a delicate balance among a variety of forces in the simulation model, which may explain that the successful folding of any alpha/beta proteins to within experimental error has yet to be reported. Here we demonstrate that it is an achievable goal to fold alpha/beta proteins with a force field emphasizing the balance between the two major secondary structures. Using our newly developed force field, we conducted extensive ab initio folding simulations on an alpha/beta protein full sequence design (FSD) employing both conventional molecular dynamics and replica exchange molecular dynamics in combination with a generalized-Born solvation model. In these simulations, the folding of FSD to the native state with high population (>64.2%) and high fidelity (C(alpha)-Root Mean Square Deviation of 1.29 A for the most sampled conformation when compared to the experimental structure) was achieved. The folding of FSD was found to follow two pathways. In the major pathway, the folding started from the formation of the helix. In the minor pathway, however, folding of the beta-hairpin started first. Further examination revealed that the helix initiated from the C-terminus and propagated toward the N-terminus. The formation of the hydrophobic contacts coincided with the global folding. Therefore the hydrophobic force does not appear to be the driving force of the folding of this protein.

| S-EPMC2780466 | biostudies-literature

Hydrophobic core formation and dehydration in protein folding studied by generalized-ensemble simulations.

Project description:Despite its small size, chicken villin headpiece subdomain HP36 folds into the native structure with a stable hydrophobic core within several microseconds. How such a small protein keeps up its conformational stability and fast folding in solution is an important issue for understanding molecular mechanisms of protein folding. In this study, we performed multicanonical replica-exchange simulations of HP36 in explicit water, starting from a fully extended conformation. We observed at least five events of HP36 folding into nativelike conformations. The smallest backbone root mean-square deviation from the crystal structure was 1.1 A. In the nativelike conformations, the stably formed hydrophobic core was fully dehydrated. Statistical analyses of the simulation trajectories show the following sequential events in folding of HP36: 1), Helix 3 is formed at the earliest stage; 2), the backbone and the side chains near the loop between Helices 2 and 3 take nativelike conformations; and 3), the side-chain packing at the hydrophobic core and the dehydration of the core side chains take place simultaneously at the later stage of folding. This sequence suggests that the initial folding nucleus is not necessarily the same as the hydrophobic core, consistent with a recent experimental phi-value analysis.

| S-EPMC2931739 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data