Studying biomolecular folding and binding using temperature-jump mass spectrometry.
Ontology highlight
ABSTRACT:
SUBMITTER: Marchand A
PROVIDER: S-EPMC6987177 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
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
Project description:We studied the microsecond folding dynamics of three beta hairpins (Trp zippers 1-3, TZ1-TZ3) by using temperature-jump fluorescence and atomistic molecular dynamics in implicit solvent. In addition, we studied TZ2 by using time-resolved IR spectroscopy. By using distributed computing, we obtained an aggregate simulation time of 22 ms. The simulations included 150, 212, and 48 folding events at room temperature for TZ1, TZ2, and TZ3, respectively. The all-atom optimized potentials for liquid simulations (OPLS(aa)) potential set predicted TZ1 and TZ2 properties well; the estimated folding rates agreed with the experimentally determined folding rates and native conformations were the global potential-energy minimum. The simulations also predicted reasonable unfolding activation enthalpies. This work, directly comparing large simulated folding ensembles with multiple spectroscopic probes, revealed both the surprising predictive ability of current models as well as their shortcomings. Specifically, for TZ1-TZ3, OPLS for united atom models had a nonnative free-energy minimum, and the folding rate for OPLS(aa) TZ3 was sensitive to the initial conformation. Finally, we characterized the transition state; all TZs fold by means of similar, native-like transition-state conformations.
| S-EPMC384698 | biostudies-literature
Project description:Temperature-jump perturbation was used to examine the relaxation kinetics of folding of the human prion protein. Measured rates were very fast (approximately 3,000 s(-1)), with the extrapolated folding rate constant at approximately 20 degrees C in physiological conditions reaching 20,000 s(-1). By a mutational analysis of core residues, we found that only 2, on the interface of helices 2 and 3, have significant phi-values in the transition state. Interestingly, a mutation sandwiched between the above 2 residues on the helix-helix contact interface had very little effect on the overall free energy of folding but led to the formation of a monomeric misfolded state, which had to unfold to acquire the native PrP(C) conformation. Another mutation that led to a marked destabilization of the native fold also formed a misfolded intermediate, but this was aggregation-prone despite the native state of this mutant being soluble. Taken together, the data imply that this fast-folding protein has a transition state that is not compact (m value analysis gives a beta(t) value of only 0.3) but contains a developing nucleus between helices 2 and 3. The fact that a mutation in this nucleus had a negligible effect on stability but still led to formation of aberrant conformations during folding implies an easily perturbed folding mechanism. It is notable that in inherited forms of human prion disease, where point mutations produce a lethal dominant condition, 20 of the 33 amino acid replacements occur in the helix-2/3 sequence.
| S-EPMC2667024 | biostudies-literature
Project description:The ability of biomolecules to fold and to bind to other molecules is fundamental to virtually every living process. Advanced experimental techniques can now reveal how single biomolecules fold or bind against mechanical force, with the force serving as both the regulator and the probe of folding and binding transitions. Here, we present analytical expressions suitable for fitting the major experimental outputs from such experiments to enable their analysis and interpretation. The fit yields the key determinants of the folding and binding processes: the intrinsic on-rate and the location and height of the activation barrier.
| S-EPMC3824551 | biostudies-literature
Project description:We provide a time- and structure-resolved characterization of the folding of the heterogeneous β-hairpin peptide Tryptophan Zipper 2 (Trpzip2) using 2D IR spectroscopy. The amide I' vibrations of three Trpzip2 isotopologues are used as a local probe of the midstrand contacts, β-turn, and overall β-sheet content. Our experiments distinguish between a folded state with a type I' β-turn and a misfolded state with a bulged turn, providing evidence for distinct conformations of the peptide backbone. Transient 2D IR spectroscopy at 45 °C following a laser temperature jump tracks the nanosecond and microsecond kinetics of unfolding and the exchange between conformers. Hydrogen bonds to the peptide backbone are loosened rapidly compared with the 5-ns temperature jump. Subsequently, all relaxation kinetics are characterized by an observed 1.2 ± 0.2-μs exponential. Our time-dependent 2D IR spectra are explained in terms of folding of either native or nonnative contacts from a common compact disordered state. Conversion from the disordered state to the folded state is consistent with a zip-out folding mechanism.
| S-EPMC3581973 | biostudies-literature
Project description:The precise mechanism of protein folding remains elusive and there is a deficiency of biophysical techniques that are capable of monitoring the individual behavior of copopulated protein conformers during the folding process. Herein, an ion mobility spectrometry (IMS) device integrated with electrospray ionization mass spectrometry (ESI-MS) has been used to successfully separate and analyze protein conformers differing in cross section and/or charge state. In an initial test, an ensemble of folded and partially folded conformers of the protein cytochrome c was separated. A detailed study undertaken on the amyloidogenic protein beta(2)-microglobulin (beta(2)m), which forms fibrils by protein unfolding followed by self-aggregation and is responsible for the disease dialysis-related amyloidosis, has generated important insights into its folding landscape. Initially, a systematic titration of beta(2)m over the pH range 2 to 7 using ESI-IMS-MS allowed individual conformers to be monitored and quantified throughout the acid denaturation process. Furthermore, a comparison of wild-type beta(2)m with single and double amino acid variants with a range of folding stabilities and propensities for amyloid fibril formation has provided illuminating evidence of the role of different conformers in protein stability and amyloidogenic aggregation. The ESI-IMS-MS data presented here not only demonstrate an important and informative further dimension to ESI-MS, but also illustrate the potential of the ESI-IMS-MS technique for unravelling protein folding enigmas in general and studying protein misfolding diseases in particular.
| S-EPMC2706321 | biostudies-literature
Project description:Arsenic (As) can form complexes with dissolved organic matter (DOM), which affects the fate of arsenic in waste sites and natural environments. It remains a challenge to analyze DOM-bound As, in particular by using a direct chromatographic separation method. Size exclusion chromatography (SEC) hyphenated with UV spectrophotometer and inductively coupled plasma mass spectrometry (ICP-MS) was developed to characterize the complexation of arsenite (As(III)) with DOM. This SEC-UV-ICP-MS method is able to differentiate As(III)-DOM complexes from free As species and has the advantage of direct determination of both free and DOM-bound As(III) through mild separation. The suitability of this method for studying As(III)-DOM complexation was demonstrated by its application, in combination with the Scatchard plot and nonlinear regression of ligand binding model, for characterizing As(III) complexation with humic acid (HA) in the absence or presence of natural sand. The results suggest that, consistent with polyelectrolytic nature of HA, the As(III)-HA complexation should be accounted for by multiple classes of binding sites. By loosely classifying the binding sites into strong (S1) and weak (S2) sites, the apparent stability constants (Ks) of the resulting As-DOM complexes were calculated as logK(s1) = 6.5-7.1 while logK(s2) = 4.7-5.0.
| S-EPMC3612379 | biostudies-literature
Project description:Crucial to revealing mechanistic details of protein folding is a characterization of the transition state ensemble and its structural dynamics. To probe the transition state of ubiquitin thermal unfolding, we examine unfolding dynamics and kinetics of wild-type and mutant ubiquitin using time-resolved nonlinear infrared spectroscopy after a nanosecond temperature jump. We observe spectral changes on two different time scales. A fast nonexponential microsecond phase is attributed to downhill unfolding from the transition state region, which is induced by a shift of the barrier due to the rapid temperature change. Slow millisecond changes arise from thermally activated folding and unfolding kinetics. Mutants that stabilize or destabilize beta strands III-V lead to a decreased or increased amplitude of the microsecond phase, indicating that the disruption or weakening of these strands occurs in the transition state. Unfolding features from microseconds to milliseconds can be explained by temperature-dependent changes of a two-dimensional free energy surface constructed by the native contacts between beta strands of the protein. In addition, the results support the possibility of an intermediate state in thermal unfolding.
| S-EPMC3319365 | biostudies-literature
Project description:Wound fluid is a complex biological sample containing byproducts associated with the wound repair process. Contemporary techniques, such as immunoblotting and enzyme immunoassays, require extensive sample manipulation and do not permit the simultaneous analysis of multiple classes of biomolecular species. Structural mass spectrometry, implemented as ion mobility-mass spectrometry (IM-MS), comprises two sequential, gas-phase dispersion techniques well suited for the study of complex biological samples because of its ability to separate and simultaneously analyze multiple classes of biomolecules. As a model of diabetic wound healing, poly(vinyl alcohol) sponges were inserted subcutaneously into nondiabetic (control) and streptozotocin-induced diabetic rats to elicit a granulation tissue response and to collect acute wound fluid. Sponges were harvested at days 2 or 5 to capture different stages of the early wound-healing process. Utilizing IM-MS, statistical analysis, and targeted ultraperformance liquid chromatography analysis, biomolecular signatures of diabetic wound healing have been identified. The protein S100-A8 was highly enriched in the wound fluids collected from day 2 diabetic rats. Lysophosphatidylcholine (20:4) and cholic acid also contributed significantly to the differences between diabetic and control groups. This report provides a generalized workflow for wound fluid analysis demonstrated with a diabetic rat model.
| S-EPMC3622049 | biostudies-literature
Project description:Methanococcus maripaludis is a methanogenic archaeon. Within its genome, there are two operons for membrane associated hydrogenases, eha and ehb. To investigate the regulation of ehb on the cell, an S40 mutant was constructed in such a way that a portion of the ehb operon was replaced by pac cassette in the wild type parental strain S2 (done by Whitman's group at the University of Georgia). The S40 and S2 strains were grown in 14N and 15N media with acetate separately. A biological replicate was made by switching the media. Mass spectrometry based quantitative proteomics were done on the mixtures to investigate the differences in expression patterns between S40 and S2. Keywords: isotope labeling mass spectrometry, quantitative proteomics
2005-09-20 | GSE2744 | GEO