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NMR技术应用于大蛋白和膜蛋白。根据Wand博士小组建立的方法，将溶剂化的泛素溶解在丁烷中，其中含有二（2-乙基己基）磺基琥珀酸钠（AOT）作为表面活性剂。将获得完整的共振分配和结构计算将进行泛素封装在反胶束。这将测试在反胶束中应用三重共振NMR技术的可行性，并且在反胶束中获得的结构与晶体结构的比较将证明并验证应用于水溶性蛋白质的方法。接下来，我们将尝试采用该方法来研究甘油二酯激酶。圆二色性和荧光方法将被用来开发的条件，这种膜蛋白在其功能形式的封装。将通过三重共振NMR方法进行二酰甘油激酶的共振归属和结构解析。如果时间允许，这种方法可以应用于配体-受体复合物。

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.