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Temperature-Jump Fluorescence Provides Evidence for Fully Reversible Microsecond Dynamics in a Thermophilic Alcohol Dehydrogenase
- Meadows, Corey W., Balakrishnan, Gurusamy, Kier, Brandon L., Spiro, Thomas
G., Klinman, Judith P.
- Journal of the American Chemical Society 2015 v.137 no.32 pp. 10060-10063
- alcohol dehydrogenase, fluorescence, fluorescence emission spectroscopy, histidine, ionization, mutants, temperature
- Protein dynamics on the microsecond (μs) time scale were investigated by temperature-jump fluorescence spectroscopy as a function of temperature in two variants of a thermophilic alcohol dehydrogenase: W87F and W87F:H43A. Both mutants exhibit a fast, temperature-independent μs decrease in fluorescence followed by a slower full recovery of the initial fluorescence. The results, which rule out an ionizing histidine as the origin of the fluorescence quenching, are discussed in the context of a Trp49-containing dimer interface that acts as a conduit for thermally activated structural change within the protein interior.