TIME-RESOLVED MAGNETIC CIRCULAR DICHROISM

本项目将应用快速时间分辨磁圆二色（TRMCD）和磁光学旋转色散（TRMORD）光谱技术研究血红素蛋白的功能和折叠。新的光学方法采用准零椭偏和偏振法来研究含有磁光活性发色团（如血红素和芳香氨基酸）的生物分子的快速动力学过程（纳秒到秒时间尺度）。功能性配体再结合研究的总体计划是研究配体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的反应性，并揭示血红素蛋白在氧化代谢中的各种功能。提出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理解这种合作系统中变构R产率T转变的动力学。我们将在纳秒和微秒时间尺度上对近紫外色氨酸带、血红素基Soret带和可见光带的TRMCD光谱进行检测，以确定红蛋白在r态羧基加合物光解后的三级和四级结构变化特征。同样，肌红蛋白中配体光解的TRMCD/MORD研究将直接用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。在细胞色素c氧化酶的铜血红素铁位点催化氧化还原反应，最终消耗细胞中的分子氧。本文提出的双金属位点配体动力学的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氧化还原化学中释放的能量偶联到沿梯度泵送的质子上？最后，在提出的另一个主要研究方向中，TRMCD/MORD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，这项工作将寻找直接的光谱动力学证据，证明有偏差的扩散动力学类型被认为是在新的能源景观观点中表征蛋白质链折叠的最早事件。

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.