X-RAY DIFFRACTION ANALYSIS OF METABOLITE SENSING RIBOSWITCHES, G-PROTEIN/SMALL M

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 该提案的目标是收集与生物学和公共卫生相关的三种不同生物大分子的高分辨率X射线衍射数据。我们的第一个目标将是了解细菌PreQ1核糖开关的代谢物识别模式。这些信息对于理解这种新的基因调控模式是必不可少的，这种模式可以作为抗菌靶标。以前，我们确定了在SSRL的PreQ1核糖开关的结构，现在已经获得了在各种代谢物存在下生长的新晶体，这些代谢物具有广泛的结合亲和力（Kd = 5 nM至500 nM）。我们还在研究G蛋白β-γ与各种小分子的结合模式，这些小分子被我们的合作者证明会影响细胞信号传导。 假设这些分子结合在β亚基上的"热点"中。 我们的目标是通过差分傅立叶技术定位这种分子，这对开发G蛋白信号转导的新型效应器具有重要意义。 最后，我们正在努力确定HIV-1蛋白质Vif与Cullin-RING连接酶复合物组分的晶体结构。这种病毒/宿主蛋白质相互作用尚未在分子水平上表征。我们的目标是识别衍射良好的晶体，以便进行结构测定。 我们的复合物包括两种人类蛋白质，EloB和EloC，以及HIV-1 Vif。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The goal of this proposal is to collect high-resolution X-ray diffraction data for three different biomacromolecules that are relevant to biology and public health. Our first objective will be to learn the mode of metabolite recognition by a bacterial, PreQ1 riboswitch. Such information is essential to understand this novel gene regulatory paradigm, which may serve as an antimicrobial target. Previously we determined the structure of the PreQ1 riboswitch at SSRL and have now obtained new crystals grown in the presence of various metabolites that span a broad range of binding affinities (Kd = 5 nM to 500 nM). We are also investigating the mode of binding of the G-protein beta-gamma to various small molecules shown to affect cell signaling by our collaborator. These molecules are hypothesized to bind in a "hotspot" on the beta subunit. Our goal is to locate this molecules by difference Fourier techniques, which has implications for developing novel effectors of G-protein signaling. Finally, we are working to determine the crystal structure of the HIV-1 protein Vif in complex with components of the Cullin-RING ligase complex. This viral/host protein interaction has not been characterized at the molecular level. Our goal is to identify well-diffracting crystals in order to proceed with a structure determination. Our complex comprises two human proteins, EloB and EloC, as well as HIV-1 Vif.