VERY HIGH RESOLUTION STRUCTURES OF THE FFH GTPASE

所有细胞都有将蛋白质定位到不同细胞隔间的机制。蛋白质与细胞膜的共翻译靶向是由信号识别颗粒(SRP)介导的，它识别新生多肽的氨基末端信号序列，并将翻译的核糖体靶向膜受体。SRP核糖核蛋白的结构和功能元件在系统发育上高度保守。该颗粒的一种蛋白质组分SRP54或FFH(在原核生物中)特别令人感兴趣，因为它提供对疏水信号肽的特异性识别，也是一种直接与膜受体相互作用的GTP酶。GTP酶是一个普遍存在的蛋白质家族，它利用GTP的结合和水解来引发各种细胞功能--研究得很好的GTP酶参与了翻译、细胞调节和信号转导。该建议旨在详细了解FFH的NG GTPase结构域的蛋白质结构。这些结果将有助于我们理解这种特定的GTP酶在SRP途径中的作用，更广泛地说，有助于我们深入了解不同的GTP酶是如何建立在共同的核心蛋白结构上来引发不同的细胞功能的。该项目有三个结晶学目标。从现有的晶体中获得高度精细的超高分辨率(1.0埃)的与apo和GDP结合的NG GTP酶的结构模型。改进现有的镁GDP型晶体，以便我们能够以类似的详细程度描述该结构。用非水解性核苷酸类似物或GTP结晶结合镁GTP的NG GTP酶，并对其晶体结构进行解析。跨越GTPase配体状态的结构数据将为详细和准确分析蛋白质、水、镁和核苷酸之间的相互作用提供基础。它们还应该揭示在较低分辨率的结构中未解决的结构现象--蛋白质立体化学的偏差、包装缺陷和构象底物--是否可能与GTPase功能有关。这些分析将涉及GTP酶的化学和蛋白质设计原则，这些原则有助于在GTP结合和水解的循环中动员不同的结构基序。

All cells contain mechanisms by which proteins are targeted to different cellular compartments. Co-translational targeting of proteins to cell membranes is mediated by the signal recognition particle (SRP), which recognizes the amino-terminal signal sequence of the nascent polypeptide and targets the translating ribosome to a membrane receptor. Structural and functional elements of the SRP ribonucleoprotein are highly phylogenetically conserved. One protein component of the particle, SRP54 or Ffh (in prokaryotes), is of particular interest because it provides specific recognition of the hydrophobic signal peptide and is also a GTPase which interacts directly with the membrane receptor. GTPases are a ubiquitous family of proteins which use the binding and hydrolysis of GTP to elicit various cellular functions - well studied GTPases are involved in translation, cell regulation, and signal transduction. This proposal is directed towards detailed protein structural understanding of the NG GTPase domain of Ffh. The results will contribute to our understanding of the role of this specific GTPase in the SRP pathway, and, more generally, give insight into how different GTPases build on a common core protein structure to elicit different cellular function. The project has three crystallographic objectives. To obtain highly refined ultra-high (1.0 Angstroms) resolution structural models of the apo- and GDP-bound NG GTPase from crystals now in hand. To improve existing MgGDP-form crystals so that we can describe that structure at a similar level of detail. And, to crystallize the MgGTP-bound NG GTPase using nonhydrolyzable nucleotide analogs or GTP, and to solve its crystal structure as well. The structural data, spanning the ligand states of the GTPase, will provide the basis for detailed and accurate analysis of the interaction between the protein, water, magnesium, and nucleotide. They should also reveal whether structural phenomena not resolved in structures at lower resolution - deviations of the protein stereochemistry, packing imperfections, and conformational substrates - may be relevant to GTPase function. The analyses will address the chemistry of the GTPase and the protein design principles which facilitate mobilization of different structural motifs during the cycle of GTP binding and hydrolysis.