PROTEIN STRUCTURE BY ANALYSIS OF SPIN LABEL INTERACTIONS

本提案的长期目标是开发和应用新的分析方法，利用EPR光谱从蛋白质的位点定向自旋标记（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。考虑到用于确定包括完整膜蛋白在内的蛋白质的结构特征和功能相关结构转变的SDSL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。在短期内，提出的工作的主要重点是开发方法来确定精确的探针间距离和两个相互作用的自旋标记位点之间的方向分布-特异性地结合到蛋白质中。所提出的方法是先前工作的逻辑扩展，这些工作已经证明了多频EPR和先进的全球分析工具对严格数据解释的重要性。将解决四个具体目标：1)将开发新的全局分析算法，用于从探针之间部分静态排序的多频EPR数据中提取探针间距离和相对方向；2)该算法将用于分析双自旋标记的螺旋肽和T4溶菌酶双SDSL突变体的实验数据。这些研究将对所制订的方法的准确性和能力提供重要的核查；3)同样的算法将用于确定α晶体蛋白和HSP 27突变体的探针间距离和相对方向，然后这些约束将用于构建假定的底物结合域和由这些蛋白形成的寡聚物种的亚基间界面的三级和四级结构模型；4)针对探针间存在动态无序的情况，开发新的全局分析算法，从多频EPR数据中提取探针间距离和平均相对方位。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，并且通过结合这些互补的结构约束，将不得不表征更少的突变体，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运动的结构转变。

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.