Crystallographic Studies of the PTS Proteins

在磷酸烯醇丙酮酸：糖磷酸转移酶系统（PTS）中所涉及的蛋白质的三维结构将由x射线晶体学确定。这种复杂的多酶系统是为数不多的运输系统之一，其成分，包括一些膜结合的成分，已经被纯化。晶体学将补充分子生物学和生物化学方面的协同效应，以研究细菌利用蛋白质磷酸化转移链调节糖跨膜运输的机制。各种蛋白质的结构测定将有助于分析蛋白质磷酸化调控的性质，以及伴随这一过程的构象转变。由于PTS是细菌独有的，因此获得的信息可能与我们利用PTS进行治疗的能力有关。在这个状态下，重点是可溶性蛋白。类似的方法将用于获取其他PTS蛋白磷酸化结构的信息。根据问题的不同，可以采用多重同构置换法或分子置换法来确定结构。为了获得更好的精度，确定的结构将被细化到尽可能高的分辨率。一旦结构可用，将设计和分析探测蛋白质-蛋白质和蛋白质-底物相互作用的突变体，并选择有趣的突变体进行结晶并确定其结构。

The three dimensional structure of the proteins involved in the phosphoenolpyruvate: sugar phosphotransferase system (PTS) will be determined by x-ray crystallography. This complex multi-enzyme system is one of the few transport systems whose components, including some of the membrane bound ones, have been purified. The crystallography will complement the molecular biology and biochemical aspects of a collaborative effect to study the mechanism by which bacteria employ a protein phosphoryl transfer chain to regulate sugar transport across the membrane. The structure determination of the various proteins will facilitate the analysis of the nature of regulation by protein phosphorylation, and the conformational transition that accompanies such processes. Since the PTS is unique to bacteria, the information obtained may pertain to our ability to take advantage of the PTS therapeutically. At this state, the focus is on the soluble proteins. Similar approaches will be used to obtain information about the phosphorylated structures of the other PTS proteins. Structure determination will be done either by the Multiple Isomorphous Replacement or by the Molecular Replacement methods, depending on the problem. For better accuracy, the determined structures will be refined to the highest resolution possible. Once structures become available, mutants that probe the protein-protein and protein-substrate interactions will be designed and analyzed biochemically, and selected interesting mutants will be crystallized and their structure determined.