Dataset Information

Activity-Related Microsecond Dynamics Revealed by Temperature-Jump Forster Resonance Energy Transfer Measurements on Thermophilic Alcohol Dehydrogenase.

ABSTRACT: Previous studies of a thermophilic alcohol dehydrogenase (ht-ADH) demonstrated a range of discontinuous transitions at 30 °C that include catalysis, kinetic isotope effects, protein hydrogen-deuterium exchange rates, and intrinsic fluorescence properties. Using the Förster resonance energy transfer response from a Trp-NADH donor-acceptor pair in T-jump studies of ht-ADH, we now report microsecond protein motions that can be directly related to active site chemistry. Two distinctive transients are observed: a slow, kinetic process lacking a temperature break, together with a faster transient that is only detectable above 30 °C. The latter establishes a link between enzyme activity and microsecond protein motions near the cofactor binding site, in a region distinct from a previously detected protein network that communicates with the substrate binding site. Though evidence of direct dynamical links between microsecond protein motions and active site bond cleavage events is extremely rare, these studies highlight the potential of T-jump measurements to uncover such properties.

SUBMITTER: Vaughn MB

PROVIDER: S-EPMC5802873 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Activity-Related Microsecond Dynamics Revealed by Temperature-Jump Förster Resonance Energy Transfer Measurements on Thermophilic Alcohol Dehydrogenase.

Vaughn Morgan B MB Zhang Jianyu J Spiro Thomas G TG Dyer R Brian RB Klinman Judith P JP

Journal of the American Chemical Society 20180111 3

Previous studies of a thermophilic alcohol dehydrogenase (ht-ADH) demonstrated a range of discontinuous transitions at 30 °C that include catalysis, kinetic isotope effects, protein hydrogen-deuterium exchange rates, and intrinsic fluorescence properties. Using the Förster resonance energy transfer response from a Trp-NADH donor-acceptor pair in T-jump studies of ht-ADH, we now report microsecond protein motions that can be directly related to active site chemistry. Two distinctive transients ar ...[more]

PMID: 29323490

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Project description:Two single-tryptophan variants were generated in a thermophilic alcohol dehydrogenase with the goal of correlating temperature-dependent changes in local fluorescence with the previously demonstrated catalytic break at ca. 30 °C (Kohen et al., Nature 1999, 399, 496). One tryptophan variant, W87in, resides at the active site within van der Waals contact of bound alcohol substrate; the other variant, W167in, is a remote-site surface reporter located >25 Å from the active site. Picosecond-resolved fluorescence measurements were used to analyze fluorescence lifetimes, time-dependent Stokes shifts, and the extent of collisional quenching at Trp87 and Trp167 as a function of temperature. A subnanosecond fluorescence decay rate constant has been detected for W87in that is ascribed to the proximity of the active site Zn(2+) and shows a break in behavior at 30 °C. For the remainder of the reported lifetime measurements, there is no detectable break between 10 and 50 °C, in contrast with previously reported hydrogen/deuterium exchange experiments that revealed a temperature-dependent break analogous to catalysis (Liang et al., Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9556). We conclude that the motions that lead to the rigidification of ht-ADH below 30 °C are likely to be dominated by global processes slower than the picosecond to nanosecond motions measured herein. In the case of collisional quenching of fluorescence by acrylamide, W87in and W167in behave in a similar manner that resembles free tryptophan in water. Stokes shift measurements, by contrast, show distinctive behaviors in which the active-site tryptophan relaxation is highly temperature-dependent, whereas the solvent-exposed tryptophan's dynamics are temperature-independent. These data are concluded to reflect a significantly constrained environment surrounding the active site Trp87 that both increases the magnitude of the Stokes shift and its temperature-dependence. The results are discussed in the context of spatially distinct differences in enthalpic barriers for protein conformational sampling that may be related to catalysis.

Dataset Information

Activity-Related Microsecond Dynamics Revealed by Temperature-Jump Forster Resonance Energy Transfer Measurements on Thermophilic Alcohol Dehydrogenase.

Publications

Activity-Related Microsecond Dynamics Revealed by Temperature-Jump Förster Resonance Energy Transfer Measurements on Thermophilic Alcohol Dehydrogenase.

Similar Datasets

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