PROTEIN STRUCTURE BY ANALYSIS OF SPIN LABEL INTERACTIONS

这项建议的长期目标是开发和应用新的分析方法，从使用EPR波谱的蛋白质的定点自旋标记(SDSL)研究中提取准确的结构信息。这些进展将为现代结构研究提供一个新的维度。鉴于用于确定蛋白质结构特征和功能相关结构转变的SDSL的数量迅速增加，所开发的方法将对现代结构生物学产生直接和普遍的影响。短期内，拟议工作的主要重点是开发确定准确的相互作用探针距离的方法，以及特定结合到蛋白质中的两个相互作用的自旋标记之间的取向分布。拟议的方法是先前工作的逻辑延伸，这些工作证明了多频率EPR和先进的全球分析工具对严格的数据解释的重要性。L)将开发新的全局分析算法，用于在探针之间部分静态排序的情况下从多频电子顺磁共振数据中提取探针间距离和相对取向；2)该新算法将用于分析双自旋标记的螺旋多肽和T4溶菌酶的双SDSL突变体的实验数据。这些研究将为所开发的方法的准确性和能力提供重要的验证；3)相同的算法将被用于确定αAcrystallin和HSP 27突变体的探针间距离和相对取向，然后这些约束将被用于构建由这些蛋白质形成的同源低聚物种的假定底物结合结构域和亚基间界面的三级和四级结构模型；4)将开发新的全局分析算法来从多频EPR数据中提取探针间距离和平均相对取向，以用于探针之间存在动态无序的情况。该项目的一般假设是，在广泛的实验条件下，可以从蛋白质结构的SDSL研究中获得准确的距离和几何信息，通过结合这些互补的结构约束，必须表征更少的突变，以便充分约束三级结构模型，并充分表征涉及蛋白质结构域移动的结构转变。

The long term goal of this proposal is to develop and apply new analytical methods for extracting accurate structural information from site-directed spin labeling (SDSL) studies of proteins using EPR spectroscopy. These advances will provide a new dimension to modem structural studies. Given the rapidly growing number of SDSL for determining structural features and functionally relevant structural transitions of proteins including integral membrane proteins, the methods developed will have an immediate and general impact on modern structural biology. 0The major emphasis of the proposed work, in the short term, is the development of methods for determining accurate interprobe distances and the distribution of orientations between two interacting spin labels site- specifically incorporated into proteins. The proposed methods are logical extensions of previous work that has demonstrated the importance of multifrequency EPR and advanced global analysis tools for rigorous data interpretation. Four Specific Aims will be addressed: l) New global analysis algorithms will be developed for extracting interprobe distance and relative orientation from multifrequency EPR data for the case of partial static ordering between the probes; 2) This new algorithm will be employed to analyze experimental data from di-spin labeled helical peptides and from double SDSL mutants of T4 lysozyme. These studies will provide an important verification of the accuracy and capabilities of the methods developed; 3) This same algorithm will be employed to determine interprobe distances and relative orientations for mutants of alphaAcrystallin and HSP 27, and these constraints will then be employed to construct tertiary and quaternary structural models for the putative substrate binding domain and the intersubunit interface of the homo- oligomeric species formed by these proteins; and 4) New global analysis algorithms will be developed to extract interprobe distance and average relative orientation from multifrequency EPR data for the case where there is dynamic disordering between the probes. The general hypothesis of this project is that accurate distance and geometry information can be obtained from SDSL studies of protein structure under a wide range of experimental conditions and that by combining these complementary structural constraints, many fewer mutants will have to be characterized in order to adequately constrain tertiary structural models and to fully characterize structural transitions which involve domain movements in proteins.