Protein Structure by EPR Distance Determination and Molecular Modeling

描述(由申请人提供)：该项目的目标是开发可用于从蛋白质的定点定向自旋标记(SDSL)研究中提取结构和动力学信息的计算工具。这些新的工具将通过扩展SDSL和电子顺磁共振(EPR)光谱的能力来确定蛋白质结构和功能动力学，从而立即产生重大影响。将开发用于直接从自旋标记蛋白质的分子动力学(MD)模拟计算连续波EPR(CW-EPR)谱的程序。将计算的线型与在一组不同环境中自旋标记的T4溶菌酶(T4L)的实验光谱进行比较，将允许详细评估MD模拟重现氮氧化物侧链的动态行为的情况。MD和蒙特卡罗(MC)模拟策略的结合将被用来预测由CW-EPR或双电子-电子共振(DER)谱测量的偶极耦合自旋标记对之间的距离分布。将评估使用低微波频率(L频段)和多频段(L、X、Q和W频段)测量CW-EPR进行距离测量的情况。低频测量有望减少两个氮氧化物相对取向的影响，从而提供更纯粹的距离分布本身的测量。旋转自由度降低的备用自旋标签将用于CW-EPR和Deer距离测量。最后，通过这些努力获得的知识将被应用于SDSL研究，即自然产生的Pro对带3蛋白(CDB3)细胞质结构域(CDB3)结构的影响。这种突变导致遗传性球形红细胞增多症和活体溶血性贫血。对CDB3基因P327R突变的研究将有助于更好地了解红细胞膜的结构以及带3突变在红细胞膜遗传性疾病中所起的作用。总的来说，本建议中开发的工具将适用于世界各地实验室中对蛋白质结构、组装和功能动力学的广泛SDSL研究。

DESCRIPTION (provided by applicant): The objectives of this project are to develop computational tools that can be used to extract structure and dynamics information from site-directed spin-labeling (SDSL) studies of proteins. These novel tools will have an immediate and significant impact by extending the capabilities of SDSL and electron paramagnetic resonance (EPR) spectroscopy for the determination of protein structures and functional dynamics. Procedures will be developed for calculating a continuous wave EPR (CW-EPR) spectrum directly from molecular dynamics (MD) simulations of a spin-labeled protein. Comparison of calculated lineshapes with experimental spectra of T4 lysozyme (T4L) spin-labeled in a set of different environments will allow a detailed evaluation of how well MD simulations reproduce the dynamic behavior of the nitroxide side chain. A combination of MD and Monte Carlo (MC) modeling strategies will be developed to predict distance distributions between pairs of dipolar coupled spin labels measured by either CW-EPR or double electron-electron resonance (DEER) spectroscopy. The use of low microwave frequency (L-band) and multifrequency (L-, X-, Q-, and W-bands) measurements for distance determination by CW-EPR will be evaluated. Low frequency measurements hold the promise of reducing the influence of the relative orientation of the two nitroxides, thus giving a more pure measure of the distance distribution itself. An alternate spin label with reduced rotational freedom will be evaluated for its utility for both CW-EPR and DEER distance measurements. Finally, the knowledge gained by these efforts will be applied in a SDSL study of the effect of a naturally occurring proline to arginine mutation at residue 327 on the structure of the cytoplasmic domain of band 3 protein (CDB3). This mutation results in hereditary spherocytosis and hemolytic anemia in vivo. The study of the P327R mutation of CDB3 will lead to a better understanding of the architecture of the red blood cell membrane and the role that mutations in band 3 play in hereditary diseases of the red blood cell membrane. In general, the tools developed in this proposal will be applicable to a wide range of SDSL studies of protein structure, assembly, and functional dynamics in laboratories worldwide.