PROTEIN STRUCTURE DETERMINATION IN REVERSE MICELLES

该项目的最终目标是进一步开发一种新的方法，有望将现代溶液核磁共振技术应用于大分子和膜蛋白。根据Wand博士团队的既定方法，溶剂化泛素将溶解在丁烷中，其中包含作为表面活性剂的二(2-乙基己基)琥珀酸磺酸钠(AOT)。将对反胶束中包裹的泛素进行完整的共振指定和结构计算。这将测试在反胶束中应用三重共振核磁共振技术的可行性，通过将反胶束中获得的结构与晶体结构进行比较，将证明并验证应用于水溶性蛋白质的方法。下一步，我们将尝试采用二酰基甘油激酶的方法。圆二色谱和荧光法将被用来开发以其功能形式包裹这种膜蛋白的条件。利用三重共振核磁共振方法对甘油二酯激酶的结构进行归属和结构鉴定。如果时间允许，这种方法可以应用于配体-受体复合体。

The ultimate goal of the project is to further develop a novel approach that will hopefully allow the application of modern solution NMR techniques to large and membrane proteins. Solvated ubiquitin will be dissolved in butane, containing sodium dis (2-ethylhexyl) sulfosuccinate (AOT) as a surfactant, according to established methods in Dr. Wand's group. Complete resonance assignments will be obtained and structure calculations will be performed on ubiquitin encapsulated in the reverse micelle. This will test the feasibility of applying triple-resonance NMR techniques in reverse micelles, and the comparison of the structure obtained in the reverse micelle with the crystal structure will demonstrate and validate the approach as applied to a water-soluble protein. Next, we will attempt to adopt the approach to diacylglycerol kinase. Circular dichroism and fluorescence methods will be used to develop conditions for the encapsulation of this membrane protein in its functional form. Resonance assignment and structural elucidation of diacylglycerol kinase will be performed by triple-resonance NMR methods. If time allows, this approach may be applied to a ligand-receptor complex.