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M Gruebele

Publications and source records attributed to M Gruebele.

14 recordsLinked to original sources
PubMed2004

Multiple probes reveal a native-like intermediate during low-temperature refolding of ubiquitin.

We investigate the refolding of ubiquitin Phe45Trp/Ile61Ala (Ub(*)I61A) in a low-temperature, high-viscosity buffer, where folding is slowed so that apparent two-state and three-state mechanisms are readily distinguishable. Ub(*)I61A forms a compact ensemble rapidly (as judged from stopped-flow, small-angle X-ray scattering) with a secondary structure signature similar to that of the native state (as judged from stopped-flow circular dichroism from 215 nm to 250 nm), but the fluorescence signature still resembles the guanidinium-denatured state. The compact ensemble forms over a range of solvent and temperature conditions. The native fluorescence signature, which requires the tryptophan residue to be packed tightly, is acquired at least 500 times more slowly. Molecular dynamics simulations at 495 K show no contraction of the backbone in ethylene glycol buffer compared to pure aqueous buffer, and no significant effect on the local backbone structure of the unfolded protein. Only at higher simulation temperature does a backbone contraction appear. Thus, it appears unlikely that the aqueous ethylene glycol buffer fundamentally changes the folding mechanism of ubiquitin. We suggest that ubiquitin forms a compact ensemble with native-like secondary structure, but without tight packing, long before the native state.

Circular Dichroism↗
PubMed2004

Vibrational energy flow and chemical reactions.

How vibrational energy flows in molecules has recently become much better understood through the joint efforts of theory, experiment, and computation. The phenomenology of energy flow is much richer than earlier thought. We now know energy flow depends on the local structure of molecular vibrational state space. The details of the theoretically predicted transition from localized vibrations to free flow, where the molecule can act as its own heat bath, are now well-established experimentally. Energy flow is a quantum diffusive process leading to nonexponential decays, also seen in experiment. The slowness of energy flow in activated molecules causes substantial deviation from statistical Rice-Ramsperger-Kassel-Marcus (RRKM) theories for low barrier rate processes, such as isomerization. Quantitative calculations of rates in those cases are now possible.

Journal Article↗
PubMed2001

The folding mechanism of a beta-sheet: the WW domain.

The folding thermodynamics and kinetics of the Pin WW domain, a three-stranded antiparallel beta-sheet, have been characterized extensively. Folding and activation free energies were determined as a function of temperature for 16 mutants, which sample all strands and turns of the molecule. The mutational phi value (Phi(m)) diagram is a smooth function of sequence, indicating a prevalence of local interactions in the transition state (TS). At 37 degrees C, the diagram has a single pronounced maximum at turn 1: the rate-limiting step during folding is the formation of loop 1. In contrast, key residues for thermodynamic stability are located in the strand hydrophobic clusters, indicating that factors contributing to protein stability and folding kinetics are not correlated. The location of the TS along the entropic reaction coordinate Phi(T), obtained by temperature-tuning the kinetics, reveals that sufficiently destabilizing mutants in loop 2 or in the Leu7-Trp11-Tyr24-Pro37 hydrophobic cluster can cause a switch to a late TS. Phi(m) analysis is usually applied "perturbatively" (methyl truncation), but with Phi(T) to quantitatively assess TS shifts along a reaction coordinate, more severe mutations can be used to probe regions of the free energy surface beyond the TS.

Amino Acid Sequence↗
PubMed2000

Mapping the transition state of the WW domain beta-sheet.

The folding kinetics of a three-stranded antiparallel beta-sheet (WW domain) have been measured by temperature jump relaxation. Folding and activation free energies were determined as a function of temperature for both the wild-type and the mutant domain, W39F, which modifies the beta(2)-beta(3) hydrophobic interface. The folding rate decreases at higher temperatures as a result of the increase in the activation free energy for folding. Phi-Values were obtained for thermal perturbations allowing the primary features of the folding free energy surface to be determined. The results of this analysis indicate a significant shift from an "early" (Phi(T)=0. 4) to a "late" (Phi(T)=0.8) transition state with increasing temperature. The temperature-dependent Phi-value analysis of the wild-type WW domain and of its more stable W39F hydrophobic cluster mutant reveals little participation of residue 39 in the transition state at lower temperature. As the temperature is raised, hydrophobic interactions at the beta(2)-beta(3) interface gain importance in the transition state and the barrier height of the wild-type, which contains the larger tryptophan residue, increases more slowly than the barrier height of the mutant.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Submicrosecond real-time fluorescence sampling: application to protein folding.

Time-resolved fluorescence detection has become a central tool in the study of protein folding. This article briefly reviews modern fluorescence techniques and then focuses on recent improvements made possible by array photomultipliers, computer-controlled data gating, and long-memory multi-channel digitizers. It is now possible to detect fluorescence wavelength profiles and/or fluorescence decay transients very cost effectively with sub-microsecond kinetic time resolution out to long times. Folding kinetics can be analyzed by singular value decomposition (SVD) or chi-analysis. The latter provides an objective method for detecting nonexponential kinetics in two-state systems.

Equipment Design↗
PubMed1999

Observation of strange kinetics in protein folding.

Highly nonexponential folding kinetics in aqueous solution have been observed during temperature jump-induced refolding of two proteins, yeast phosphoglycerate kinase and a ubiquitin mutant. The observations are most easily interpreted in terms of downhill folding, which posits a heterogeneous ensemble of structures en route to the folded state. The data are also reconciled with exponential kinetics measured under different experimental conditions and with titration experiments indicating cooperative folding.

Amino Acid Substitution↗
PubMed1999

Observation of the nu(6) + nu(9) Band of Ketene via Resonant Coriolis Interaction with nu(8).

We observed and analyzed a b-axis Coriolis resonance between higher J states of the nu(6) + nu(9) combination band and the nu(8) fundamental of ketene in the spectral region 940-970 cm(-1). The interaction resonantly couples K(a) = 1 states of the combination band to K(a) = 0 states of the fundamental and also affects K(a) = 1, 2 states in the fundamental. Due to the involvement of strongly asymmetry-split low K levels, the rotational constants and band origin of nu(6) + nu(9) could be accurately determined and are discussed in the light of high-quality anharmonic force fields. The Coriolis coupling parameter, zeta(b)(8,6+9), is very precisely determined. A smaller perturbation, which could not be fully analyzed, is tentatively attributed to K(a) = 2 upper states in the nu(5) + nu(9) band. Copyright 1999 Academic Press.

Journal Article↗
PubMed1999

The fast protein folding problem.

During protein folding, many of the events leading to secondary and tertiary structure occur in milliseconds or faster. Modern nuclear magnetic resonance and laser detection techniques, coupled with fast initiation of the folding reaction, are probing these events in great detail. Theory, ranging from analytical models to molecular dynamics calculations, is beginning to match up with experiment. As a result, timescales, from such elementary steps as the addition of a residue to a helix to strange kinetics of collapsing protein backbones, can now be measured and interpreted.

Journal Article↗
PubMed1998

The vibrational energy flow transition in organic molecules: theory meets experiment.

Most large dynamical systems are thought to have ergodic dynamics, whereas small systems may not have free interchange of energy between degrees of freedom. This assumption is made in many areas of chemistry and physics, ranging from nuclei to reacting molecules and on to quantum dots. We examine the transition to facile vibrational energy flow in a large set of organic molecules as molecular size is increased. Both analytical and computational results based on local random matrix models describe the transition to unrestricted vibrational energy flow in these molecules. In particular, the models connect the number of states participating in intramolecular energy flow to simple molecular properties such as the molecular size and the distribution of vibrational frequencies. The transition itself is governed by a local anharmonic coupling strength and a local state density. The theoretical results for the transition characteristics compare well with those implied by experimental measurements using IR fluorescence spectroscopy of dilution factors reported by Stewart and McDonald [Stewart, G. M. & McDonald, J. D. (1983) J. Chem. Phys. 78, 3907-3915].

Journal Article↗
PubMed1998

Intramolecular vibrational dephasing obeys a power law at intermediate times.

Experimental intramolecular vibrational dephasing transients for several large organic molecules are reanalyzed. Fits to the experimental data, as well as full numerical quantum calculations with a factorized potential surface for all active degrees of freedom of fluorene indicate that power law decays, not exponentials, occur at intermediate times. The results support a proposal that power law decays describe vibrational dephasing dynamics in large molecules at intermediate times because of the local nature of energy flow.

Journal Article↗
PubMed1998

Satisfying turns in folding transitions.

Protein engineering studies show that conformations in the folding transition state ensemble can be structurally polarized. In two SH3 beta-sheet domains, the formation of hydrophobic contacts goes hand in hand with the formation of the solvated distal loop beta-turn, while large parts of the molecule remain unstructured in the ensemble.

Evolution, Molecular↗
PubMed1996

Direct observation of fast protein folding: the initial collapse of apomyoglobin.

The rapid refolding dynamics of apomyoglobin are followed by a new temperature-jump fluorescence technique on a 15-ns to 0.5-ms time scale in vitro. The apparatus measures the protein-folding history in a single sweep in standard aqueous buffers. The earliest steps during folding to a compact state are observed and are complete in under 20 micros. Experiments on mutants and consideration of steady-state CD and fluorescence spectra indicate that the observed microsecond phase monitors assembly of an A x (H x G) helix subunit. Measurements at different viscosities indicate diffusive behavior even at low viscosities, in agreement with motions of a solvent-exposed protein during the initial collapse.

Animals↗