STUDY OF TRANSIENT LIGHT-INITIATED DYNAMICS IN CIRCADIAN CLOCK PROTEINS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 在与起重机合作，我们将应用时间分辨二维FT ESR的电子转移（ET）和动物1型隐花色素（dCRY），特别是果蝇CRY（dCRY），夹带昼夜节律的激活过程的研究。时间分辨ESR光谱已被应用于CRY蛋白的不同亚类，称为CRY_DASH，但目前还没有直接测量的自由基对（RP）已在动物1型脑中观察到。Trp残基在1型β中的独特表达以及C-末端的参与表明光诱导的ET过程在该亚类中可能是独特的，并且与功能偶联。在瞬态吸收实验中，Trp自由基的光信号在光激发后在ps时间尺度上形成，但持续更长时间（> nsec）。在CRY DASH蛋白质中，RP的持续时间为微秒。 在dCRY中，（黄素腺嘌呤二核苷酸）的阴离子半醌形式FAD [FAD+-]在蓝光激发下形成，即使在氧气存在下也能稳定数分钟。自由基对的另一半，W++最初形成，但在更快的时间尺度（~微秒）上被去质子化和还原。到MS）。最初的电荷分离[FAD+- W++]可能导致ET事件的级联，伴随着蛋白质结构的变化，可能产生几种中间体，例如RNR或PS-II。 而FAD+-可以使用CW ESR检测到，在这个RP和蛋白质的演变早期事件需要仔细研究的事件发展的纳秒到毫秒的时间尺度。这可以通过使用时间分辨的2D-FT ESR来实现，其提供了所需的灵敏度和足够的时间分辨率。

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.