Toward a High Resolution Structure of an Olfactory Receptor

描述(由申请人提供)：气味检测和信号转导发生在纤毛中，它们是由气味分子与G蛋白偶联嗅觉受体相互作用而启动的。嗅觉受体组成了一个多基因大家族；在人类中，估计有400个功能基因存在于许多不同的染色体位置。在过去的几年里，关于嗅觉转导中的第二信使兴奋级联和信号终止，已经收集了大量的信息。然而，对于这一过程的第一步，即气味分子与受体本身的相互作用，人们知之甚少。这主要是由于缺乏关于嗅觉感受器的生化特性和结构的实验数据。我们研究计划的长期目标是使用三维(3D)X射线结晶学技术在原子水平上确定嗅觉受体的结构，以了解气味如何与这些专门的受体相互作用。即使只有一个结构在手，也将为利用同源建模研究其他气味受体开辟许多可能性。因此，这项R21应用的目标是：(1)使用可诱导的异源过表达系统来生产毫克量的选定嗅觉受体，(2)开始在室内测试这些蛋白质的结晶，并使用布法罗大学Hauptman-Woodward医学研究所的设施。这项拟议的研究具有重要意义，因为从这项工作中预期的探索性数据将为确定原型OR的原子结构开辟道路。通过首次提供气味分子/受体相互作用的结构基础，实现这一目标将对化学感官领域产生重大影响。这将提供一个机会，在分子水平上理解和解释使用功能分析的诱变实验的结果，无论是已公布的还是未来的。此外，原子结构将允许创建更准确的同源模型，提高预测嗅觉感受器气味结合特性的能力。这些信息可以用来设计分子，使其更紧密地结合在一起，或者抑制天然气味的结合。它可以帮助理解个体之间的嗅觉差异，这可能是由于嗅觉受体氨基酸序列的多态引起的。最后，嗅觉受体结构将对涉及G蛋白偶联受体超家族的所有学科产生重大影响，因为目前只有一种已知结构(即视紫红质)。与公共卫生相关：人们越来越需要了解荷尔蒙或气味等分子与其膜蛋白受体选择性结合的结构基础。这对于合理的药物设计和分子检测器的生产尤为重要。这项拨款旨在阐明嗅觉受体的三维结构，嗅觉受体是一大家族蛋白质，可以检测到具有不同分子结构的各种分子。

DESCRIPTION (provided by applicant): Odor detection and signal transduction occur in cilia where they are initiated by the interaction of odorant molecules with G-protein coupled olfactory receptors. Olfactory receptors comprise a large multigene family; in humans there are an estimated 400 functional genes found in many different chromosomal locations. A great deal of information has been gathered regarding both the second messenger excitation cascade in olfactory transduction and signal termination over the past years. Nonetheless, there is a paucity of knowledge about the first step in the process, namely, the interaction of an odorant molecule with the receptor, itself. This is primarily due to the lack of experimental data on the biochemical properties and structure of olfactory receptors. The long-term goal of our research program is to determine the structure of olfactory receptors at the atomic level using three-dimensional (3D) X-ray crystallographic techniques to understand how odorants interact with these specialized receptors. Having even one structure in hand would open up many possibilities to study other odorant receptors using homology modeling. Consequently, the objectives of this R21 application are to: (1) use an inducible heterologous over-expression system to produce milligram quantities of selected olfactory receptors and (2) begin assaying these proteins for crystallization in house and using the facility at Hauptman-Woodward Medical Research Institute at the University of Buffalo. The proposed research is significant because the exploratory data expected from this work would open the way to determine the atomic structure of a prototypical OR. Achieving this goal would have a major impact on the Chemical Senses field by providing for the first time the structural basis of odorant molecule/receptor interactions. This would provide an opportunity to understand and interpret at the molecular level the results from mutagenesis experiments, both published and future, that are using functional assays. Moreover, an atomic structure would allow the creation of more accurate homology models, improving the ability to predict an olfactory receptor's odorant binding properties. This information could be used to design molecules to bind more tightly or to inhibit binding of natural odorants. It could help understand variation in olfactory sensation between individual people potentially arising from olfactory receptor amino acid sequence polymorphisms. Finally, an olfactory receptor structure would have significant impact for all disciplines that involve G-protein coupled receptor superfamily, since at present there is only one known structure (i.e. rhodopsin). PUBLIC HEALTH RELEVANCE: There is a growing demand to understand the structural basis for the selective binding of molecules like hormones or odorants to their membrane protein receptors. This is particularly important for rational drug design and producing molecular detectors. This grant is aimed at elucidating the three dimensional structure of olfactory receptors, a large family of proteins that detect a wide variety of molecules with diverse molecular structures.