VERY HIGH RESOLUTION STRUCTURES OF THE FFH GTPASE

FFH GTPASE 的极高分辨率结构

• 批准号: 6351259
• 负责人: Douglas M. Freymann
• 金额: $ 24.29万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6351259
• 关键词: X ray crystallography; bioimaging /biomedical imaging; conformation; crystallization; enzyme structure; guanosine triphosphate; guanosinetriphosphatases; intermolecular interaction; physical model; protein sequence; protein structure function; ribonucleoproteins; stereochemistry; structural biology; water

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.

所有的细胞都含有将蛋白质定向到不同的细胞区室的机制。信号识别粒子（signal recognition particle， SRP）介导了蛋白质对细胞膜的共翻译靶向，SRP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到膜受体。SRP核糖核蛋白的结构和功能元件在系统发育上是高度保守的。该颗粒的一种蛋白质成分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定识别，也是一种直接与膜受体相互作用的GTPase。GTP酶是一种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- GTP酶参与翻译，细胞调节和信号转导。这一建议是针对Ffh的NG GTPase结构域的详细蛋白质结构的理解。这些结果将有助于我们理解这种特定的GTPase在SRP途径中的作用，并且更广泛地说，可以深入了解不同的GTPase如何建立在共同的核心蛋白结构上，从而引发不同的细胞功能。该项目有三个晶体学目标。从现在的晶体中获得高度精细的超高（1.0埃）分辨率的载脂蛋白和gdp结合的NG GTPase结构模型。为了改进现有的mggdp晶体，这样我们就可以在相似的细节水平上描述这种结构。并且，利用非水解核苷酸类似物或GTP来结晶mggtp结合的NG GTPase，并求解其晶体结构。这些结构数据跨越了GTPase的配体状态，将为详细准确地分析蛋白质、水、镁和核苷酸之间的相互作用提供基础。他们还应该揭示在低分辨率结构中无法解决的结构现象-蛋白质立体化学偏差，包装缺陷和构象底物-是否可能与GTPase功能有关。分析将涉及GTP酶的化学性质和蛋白质设计原则，这些原则有助于在GTP结合和水解循环中调动不同的结构基序。