Project 2/Dev't and evaluation of computational tools for structural studies by E

该项目的目标是开发和评估新的分子建模和计算方法。 从定点定向自旋标记(SDSL)研究中提取准确的结构信息。组合在一起 SDSL和电子顺磁共振波谱(EPR)的研究在以下方面取得了显著进展 过去15年(回顾见Hubbell和Altenbach，1994；Hustedt和Beth，1999；Hubbell等人，2000； Mchaourab和Perozo，2000；Columbus和Hubbell，2002；Klug和Feix，2005)，现在被广泛使用 为了研究蛋白质和大分子组装体的结构和结构转变， 其中许多化合物难以通过X射线结晶学等技术进行结构表征 和核磁共振。即使在那些可以获得原子分辨率结构的情况下，SDSL也越来越多地 在补充性研究中用来阐明决定生物的结构转变的动力学 函数(例如，董等人，2005年)。此外，还利用SDSL研究了大分子的组装 由元素组成的络合物，其各自的结构已由核磁共振或X射线确定 结晶学方法(例如Park等人，2006年)。 我们建议将现代分子动力学和计算方法的全部力量结合在一起 Lybrand实验室使用先进的EPR方法在Hustedt实验室开发工具，将大大 提高从SDSL数据获得的结构模型的质量。分子动力学(MD) 并将开发蒙特卡罗(MC)建模策略来处理 甲硫磺酸自旋标记物(MTSSL)。这些策略将被用来预测连续波 单标蛋白质的EPR(CW-EPR)线型及其距离分布 以及双标记蛋白质的双电子-电子共振(DER)实验。这些新的 计算算法将在研究良好的蛋白质T4溶菌酶(T4L)上进行测试，然后用于研究 精氨酸突变对细胞质区结构和动力学的影响 红细胞阴离子交换蛋白，带3(CDB3)，正在与本赠款的项目2合作 求婚..以下具体目标的总体目标是使结构和动态信息达到最大 从一组给定的EPR数据中导出。

The objectives of this project are to develop and evaluate novel molecular modeling and computational approaches to extract accurate structural information from site-directed spin-labeling (SDSL) studies. The combination of SDSL and electron paramagnetic resonance spectroscopy (EPR) has made remarkable advances in the past 15 years (for reviews see (Hubbell and Altenbach, 1994; Hustedt and Beth, 1999; Hubbell et al., 2000; Mchaourab and Perozo, 2000; Columbus and Hubbell, 2002; Klug and Feix, 2005) and it is now widely employed to study the structures and structural transitions of proteins and large macromolecular assemblies, many of which have been refractory to structural characterization by techniques such as X-ray crystallography and NMR. Even in those cases where atomic resolution structures can be obtained, SDSL is being increasingly employed in complementary studies to elucidate the dynamics of structural transitions that determine biological function (e.g. (Dong et al., 2005). In addition, SDSL has been employed to study the assembly of macromolecular complexes composed of elements whose individual structures have been determined by NMR or X-ray crystallographic methods (e.g. (Park et al., 2006)). We propose to combine the full power of modern molecular dynamics and computational approaches in the Lybrand laboratory with advanced EPR methods in the Hustedt laboratory to develop tools that will dramatically improve the quality of the structural models that are obtained from SDSL data. Molecular dynamics (MD) and Monte Carlo (MC) modeling strategies will be developed to treat the structure and dynamics of the methanethiosulfonate spin label (MTSSL). These strategies will be used to predict both the continuous wave EPR (CW-EPR) lineshapes of singly labeled proteins and the distance distributions measured by both CWEPR and double electron-electron resonance (DEER) experiments on doubly labeled proteins. These new computational algorithms will be tested on the well-studied protein T4 lysozyme (T4L) and then used to investigate the effect of a proline to arginine mutation on the structure and dynamics of the cytoplasmic domain of the erythrocyte anion exchange protein, band 3 (CDB3) in an ongoing collaboration with Project 2 of this grant proposal.. The overall goal of the following Specific Aims is to enable the maximum structural and dynamic information to be derived from a given set of EPR data.