TIME-RESOLVED MAGNETIC CIRCULAR DICHROISM

该项目将应用快速时间分辨磁性圆二色谱(TRMCD)和磁光旋转色散(TRMORD)光谱技术来研究血红素蛋白的功能和折叠。新的光学方法使用准零椭圆偏振法和旋光法来研究生物分子中的快速动力学过程(纳秒到秒的时间尺度)，这些生物分子含有磁光活性的生色团，如血红素和芳香族氨基酸。功能配基重新结合研究的总体方案是研究配基光解后的蛋白质松弛，目的是了解蛋白质结构如何调节血红素假体基团的反应性，并引发血红素蛋白质在氧化代谢中执行的各种功能。建议的氧转运蛋白血红蛋白动力学中间体的TRMCD研究的目的是更好地了解这种合作体系中变构R产率T跃迁的动力学。将在纳秒和微秒时间尺度上检查近紫外色氨酸带和基于血红素的Soret和可见光带的TRMCD光谱，这是球蛋白在R-态羧基加合物光解后的三级和四级结构变化的特征。同样，TRMCD/MORD对肌红蛋白中配体光解的研究将致力于解决关于这种储氧蛋白功能背后的血红素口袋动力学的悬而未决的问题。在细胞中，分子氧最终被细胞色素c氧化酶中铜-血红素铁位催化的氧化还原反应所消耗。这里提出的双金属配基动力学的TRMCD研究最终解决了生物物理学中的一个主要难题：细胞色素氧化酶如何将在这种氧化还原化学中释放的能量与质子向梯度泵送相耦合？最后，在提出的另一个主要研究方向，TRMCD/MOD技术将用于监测血红素蛋白折叠反应中的超快(亚毫秒)事件。特别是，这项工作将为有偏见的扩散动力学类型寻找直接的光谱动力学证据，这些扩散动力学被认为是从新的能量景观的观点来表征蛋白质链折叠中的最早事件。

This project will apply techniques for fast time-resolved magnetic circular dichroism (TRMCD) and magnetic optical rotatory dispersion (TRMORD) spectroscopies to the study of function and folding in heme proteins. The novel optical methods employed use quasi-null ellipsometry and polarimetry to study rapid kinetic processes (nanosecond to second time scales) in biomolecules that contain magneto-optically active chromophores such as heme and the aromatic amino acids. The overall program of the functional ligand-rebinding studies is to investigate protein relaxation after ligand photolysis with the goal of understanding how protein structure modulates the reactivity of the heme prosthetic group and elicits the variety of functions that heme proteins perform in oxidative metabolism. The proposed TRMCD studies of kinetic intermediates in the oxygen transport protein hemoglobin have the goal of better understanding the dynamics of the allosteric R yields T transition in this cooperative system. The TRMCD spectra of the near-UV tryptophan bands and the heme-based Soret and visible bands will be examined for MCD transients on the nanosecond and microsecond timescales that are characteristic of globin tertiary and quaternary structural changes after photolysis of the R-state carboxy adduct. Similarly, TRMCD/MORD studies of ligand photolysis in myoglobin will be directed toward resolving outstanding questions about the heme pocket dynamics underlying the function of this oxygen storage protein. Molecular oxygen is ultimately consumed in cells by redox reactions catalyzed at a copper-heme iron site in the enzyme cytochrome c oxidase. The TRMCD studies of ligand dynamics at the bimetallic site proposed here are ultimately addressed at a major puzzle in biophysics: How does cytochrome oxidase couple the energy released in this redox chemistry to the pumping of protons against a gradient? Finally, in the other major direction of investigation proposed, TRMCD/MORD techniques will be used to monitor ultrafast (submillisecond) events in the folding reactions of heme proteins. In particular, this work will look for direct spectrokinetic evidence for the type of biased diffusional dynamics thought to characterize the earliest events in the folding of protein chains in the new, energy landscape point of view.