PROTEIN STRUCTURE BY ANALYSIS OF SPIN LABEL INTERACTIONS

通过自旋标签相互作用分析蛋白质结构

• 批准号: 6636381
• 负责人: ERIC J HUSTEDT
• 金额: $ 15.1万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6636381
• 关键词: binding sites; bioimaging /biomedical imaging; chemical models; crystallins; electron spin resonance spectroscopy; heat shock proteins; lysozyme; model design /development; protein structure; structural biology; technology /technique development

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.

该提案的长期目标是开发和应用新的分析方法，用于使用EPR光谱从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SDSL用于确定蛋白质（包括膜蛋白）的结构特征和功能相关结构转变的数量迅速增加，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。在短期内，所提出的工作的主要重点是开发用于确定精确的探针间距离和两个相互作用的自旋标记位点特异性地掺入蛋白质之间的取向分布的方法。所提出的方法是以前的工作，证明了多频EPR和先进的全球分析工具，严格的数据解释的重要性的逻辑扩展。本论文的主要目的是：1）在探针间部分静态有序的情况下，从多频EPR数据中提取探针间的距离和相对取向; 2）将该算法应用于双自旋标记的螺旋肽和T4溶菌酶双SDSL突变体的实验数据分析。这些研究将对所制定的方法的准确性和能力提供重要的验证; 3）该相同的算法将用于确定α-腺苷酸和HSP 27的突变体的探针间距离和相对取向，然后，这些约束将用于构建推定的底物结合结构域和同源物的亚基间界面的三级和四级结构模型。这些蛋白质形成的寡聚物物种;和4）将开发新的全局分析算法，以从多频率EPR数据中提取探针间距离和平均相对取向，用于探针之间存在动态无序的情况。该项目的一般假设是，准确的距离和几何信息可以从SDSL研究蛋白质结构在广泛的实验条件下，通过结合这些互补的结构约束，少得多的突变体将不得不进行表征，以充分约束三级结构模型和充分表征结构转换，涉及蛋白质中的结构域运动。