An instrument for time resolved monitoring of fast chemical transitions: Application to the kinetics of refolding of a globular protein

Vladimir Ratner, Elisha Haas

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    16 Scopus citations


    The dynamics of kinetic intermediates of protein folding can be studied by time resolved measurements of nonradiative excitation energy transfer, in site-specific labeled protein derivatives, combined with fast mixing experiments. A new device based on the single pulse approach was developed. This experiment is performed over two time scales: the "chemical time scale" of the conformational changes (milliseconds), defined by the rates of changes of conformations in the sample, and the "spectroscopic time scale" (nanoseconds) defined by the lifetimes of the excited states of the fluorescent probes. The chemical process was synchronized by means of a fast mixing stopped-flow device. The low cost laser used here is suitable for use with dyes with excitation wavelengths of 337 nm and higher. Up to 20 fluorescence decay curves per second, can be measured within a single stopped flow run. Each fluorescence decay curve is recorded within 2.50 ns or more. The time resolution (of the spectroscopic time scale) was 0.5 ns. The noise level is low enough to estimate distance distributions from energy transfer experiments, provided that the shortest changeable lifetime component of the fluorescence decay of the donor probes would not be lower than ∼4 ns. The amount of double labeled protein which should be used for each experiment in order to obtain a full data set, with time resolution of 10 ms during protein transition, is only fourfold more than the amount needed for a stopped flow study with steady state fluorescence monitoring. The results obtained for refolding of α-lactalbumin in the presence of 1,8-anilino-naphthalene sulfonic acid from the GuHCl induced denatured state, support the model in which the probe has two states. The first state, is characterized by a fluorescence lifetime of 14.2 ±+0.5 ns and the second by a fluorescence lifetime of 0.5±0.4 us or less. During refolding the dye is transfered from the first state to the second, at a rate that coincides with the rate of the steady state flourescence change. Refolding from the acid-induced molten globule state shows a more complex change in the parameters of the fluorescence decay, probably due to partial aggregation of the protein during refolding.

    Original languageEnglish
    Pages (from-to)2147-2154
    Number of pages8
    JournalReview of Scientific Instruments
    Issue number5
    StatePublished - May 1998


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