Protein Structure by EPR Distance Determination and Molecular Modeling

通过 EPR 距离测定和分子建模确定蛋白质结构

• 批准号: 7302510
• 负责人: ERIC J HUSTEDT
• 金额: $ 27.63万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-15 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7302510
• 关键词: Affect; Anion Exchangers (Proteins); Architecture; Arginine; Behavior; Binding Proteins; Cell membrane; Computing Methodologies; Condition; Coupled; Cytoplasmic Tail; Data; Electron Spin Resonance Spectroscopy; Electrons; Environment; Erythrocyte Anion Exchange Protein 1; Erythrocytes; Evaluation; Freedom; Frequencies; Goals; Helix (Snails); Hemolytic Anemia; Hereditary Disease; Hereditary Spherocytosis; Knowledge; Label; Laboratories; Lead; Measurement; Measures; Modeling; Molecular Structure; Muramidase; Mutation; Play; Pliability; Procedures; Proline; Protein Dynamics; Proteins; Range; Relative (related person); Research Personnel; Role; Side; Site; Spectrum Analysis; Spin Labels; Structural Models; Structure; Surface; Testing; Time; band 3 protein Tuscaloosa; computerized tools; ear helix; in vivo; microwave electromagnetic radiation; molecular dynamics; molecular modeling; novel; programs; protein function; protein protein interaction; protein structure; research study; tool

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.

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位点定向自旋标记（SDSL）研究中提取结构和动力学信息的计算工具。这些新工具将通过扩展SDSL和电子顺磁共振（EPR）光谱测定蛋白质结构和功能动力学的能力，产生直接和重大的影响。程序将开发计算连续波EPR （CW-EPR）光谱直接从分子动力学（MD）模拟自旋标记的蛋白质。将计算的线形与T4溶菌酶（T4L）在一组不同环境中自旋标记的实验光谱进行比较，可以详细评估MD模拟如何很好地再现氮氧化物侧链的动态行为。将开发MD和蒙特卡罗（MC）建模策略的结合，以预测偶极耦合自旋标签对之间的距离分布，这些自旋标签是由CW-EPR或双电子-电子共振（DEER）光谱测量的。使用低微波频率（L-波段）和多频率（L-, X-， Q-和w -波段）测量距离确定CW-EPR将进行评估。低频测量有望减少两种氮氧化物相对取向的影响，从而对距离分布本身进行更纯粹的测量。减少旋转自由度的替代自旋标签将评估其在CW-EPR和DEER距离测量中的效用。最后，通过这些努力获得的知识将应用于SDSL研究中，研究天然存在的脯氨酸到精氨酸突变对带3蛋白（CDB3）细胞质结构域结构的影响。这种突变导致遗传性球形红细胞增多症和体内溶血性贫血。对CDB3 P327R突变的研究将有助于更好地了解红细胞膜的结构以及3带突变在红细胞膜遗传性疾病中的作用。总的来说，本提案中开发的工具将适用于全球实验室中广泛的蛋白质结构，组装和功能动力学的SDSL研究。