STUDY OF TRANSIENT LIGHT-INITIATED DYNAMICS IN CIRCADIAN CLOCK PROTEINS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. In collaboration with Crane we will apply time-resolved 2D-FT ESR to the study of electron transfer (ET) and activation processes of animal type-1 cryptochromes (CRYs), in particular the Drosophila CRY (dCRY) that entrains circadian rhythms. Time-resolved ESR spectroscopy has been applied to a different subclass of CRY proteins, called CRY_DASH, but currently no direct measurement of radical pairs (RP's) have been observed in animal type 1 CRYs. The unique arrangment of Trp residues in type 1 CRYs, and the involvement of the C-terminus suggests that photoinduced ET processes may be unique in this subclass, and coupled to function. In transient absorption experiments, optical signals for Trp radicals form on the ps time scale following light excitation, but persist much longer (> nsec). In CRY DASH proteins the RP's persist for microseconds. In dCRY, the anionic semiquinone form of (flavine adenine dinucleotide), FAD [FAD+-] forms on blue light excitation and is stable for minutes even in the presence of oxygen. The other half of the radical pair, W++ forms initially but is deprotonated and reduced on a much faster timescale (~microsec. to ms). The initial charge separation [FAD+- W++] could result in a cascade of ET events with concomitant changes to protein structure, possibly producing several intermediates, as in the cases for example of RNR or PS-II. Whereas FAD+- can be detected using CW ESR, early events in the evolution of this RP and the protein require a careful study of the events developing on the nanosecond to millisecond time scale. This can be accomplished by using time-resolved 2D-FT ESR, which provides the required level of sensitivity and adequate time resolution.