RUI: NMR Investigation of the Role of Protein Dynamics in the Selective Interactions between RGS and Galpha Signaling Proteins

RUI：蛋白质动力学在 RGS 和 Galpha 信号蛋白选择性相互作用中作用的 NMR 研究

• 批准号: 1158177
• 负责人: Karin Crowhurst
• 金额: $ 43.61万
• 依托单位: The University Corporation, Northridge
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1158177&HistoricalAwards=false
• 关键词: RUI; NMR; Investigation; Role; Protein

Intellectual Merit. Proteins are complex molecules that are the workhorses of biological organisms: they catalyze reactions, carry nutrients, provide structural support and transmit signals, just to name a few examples. The molecular structure of a protein is composed of a long string (polymer) of linked amino acids, with a variety of side chains that extend out from the backbone (similar to a charm bracelet). This string is then folded into a specifically-defined 3-dimensional structure that constitutes its functional form. Many critical signal transmission pathways are initiated by the activities of Galpha protein subunits, which are in turn controlled by regulators of G-protein signaling (RGS proteins). In particular, interactions between RGS proteins and their specific G proteins turn on and off pathways that influence brain development and function. Although much has been learned about the mechanisms of signal transmission, more research is required to obtain a thorough, molecular-level grasp of brain function. This research project focuses on two of the 20 proteins in the RGS protein family: RGS4 and RGS7. Both proteins can control the signaling of Gialpha1 (a common type of Galpha protein subunit), but the interaction between RGS4 and Gialpha1 is ~60 times stronger than that between RGS7 and Gialpha1. Normally, these differences are explained by variations in the types of amino acids that are found in the interface between the two proteins: one pair of interacting proteins may have more compatible and therefore stronger contacts than the other pair. However, when looking at the interfaces of RGS4 and RGS7 with Gialpha1, there is only one small difference between them and it cannot account for the large difference in binding preference.Other researchers have found evidence for changes in the three-dimensional structures of the protein backbones and/or the flexibility of the molecules in both RGS and Gialpha1 proteins when they interact. In addition, scientists have recently become more aware that these kinds of motions can have powerful influences over the functional capabilities of signaling proteins. This research therefore tests the hypothesis that internal movements within RGS proteins play critical roles in both their abilities to interact with various types of Galpha protein subunits and also their different degrees of preference. In order to investigate this we are using a nuclear magnetic resonance (NMR) spectrometer, which is a very powerful instrument that permits the detailed analysis of proteins; in fact, we can watch the activities of individual atoms within these molecules and thereby gain in-depth insight into their activities. The results from this work will fundamentally improve our awareness of the role of protein motions in molecular recognition and help explain the selectivity of RGS proteins for particular Galpha protein subunits, and accelerate advancements in our overall understanding of signaling mechanisms and control at the molecular level.Broader impacts. California State University, Northridge (CSUN) is a primarily undergraduate institution. Over half of the science students are from underrepresented minority groups; many students are the first in their family to attend college and are economically disadvantaged. There are few minority scientists doing protein biophysical research; this project should help to expand those numbers by drawing from the large pool of underrepresented students in the department. This project will also provide unprecedented opportunities to CSUN students to participate in cutting-edge research using modern methods and technology. For many students this will be the first (and possibly only) chance they will have to experience real research challenges and to learn troubleshooting and problem-solving skills. These new research skills will translate into increased self-confidence, improved learning in courses, and an increased likelihood that they will be inspired to pursue advanced degrees in Biochemistry or Structural Biology. Speaking more generally, a third major outcome of the project will be to illustrate to the broader public that high-quality, impactful research can be performed at a primarily undergraduate institution.

智力优势。蛋白质是复杂的分子，是生物有机体的主力：它们催化反应，携带营养物质，提供结构支持和传递信号，仅举几个例子。蛋白质的分子结构由一长串（聚合物）连接的氨基酸组成，具有从主链延伸出来的各种侧链（类似于魅力手镯）。然后将该弦折叠成构成其功能形式的特定定义的三维结构。许多关键的信号传递途径是由G α蛋白亚基的活性启动的，而G α蛋白亚基的活性又受G蛋白信号调节因子（RGS蛋白）的控制。特别是，RGS蛋白与其特异性G蛋白之间的相互作用开启和关闭影响大脑发育和功能的途径。虽然我们已经对信号传递的机制有了很多了解，但还需要更多的研究来彻底掌握大脑功能的分子水平。该研究项目的重点是RGS蛋白家族20种蛋白中的两种：RGS 4和RGS 7。这两种蛋白质都可以控制Gialpha 1（一种常见的Galpha蛋白亚基）的信号传导，但RGS 4和Gialpha 1之间的相互作用比RGS 7和Gialpha 1之间的相互作用强约60倍。通常情况下，这些差异是由两种蛋白质之间界面上发现的氨基酸类型的变化来解释的：一对相互作用的蛋白质可能比另一对具有更相容的接触，因此接触更强。然而，当观察RGS 4和RGS 7与Gialpha 1的界面时，它们之间只有一个很小的差异，无法解释结合偏好的巨大差异。其他研究人员发现了蛋白质骨架的三维结构变化的证据和/或RGS和Gialpha 1蛋白相互作用时分子的柔性。此外，科学家们最近越来越意识到，这些运动可以对信号蛋白的功能产生强大的影响。因此，这项研究测试了RGS蛋白内部运动在其与各种类型的Galpha蛋白亚基相互作用的能力以及其不同程度的偏好中发挥关键作用的假设。为了研究这一点，我们使用核磁共振（NMR）光谱仪，这是一种非常强大的仪器，可以对蛋白质进行详细分析;事实上，我们可以观察这些分子中单个原子的活动，从而深入了解它们的活动。这项工作的结果将从根本上提高我们对蛋白质运动在分子识别中的作用的认识，并有助于解释RGS蛋白对特定Galpha蛋白亚基的选择性，并加速我们在分子水平上对信号机制和控制的整体理解的进步。加州州立大学北岭分校（CSUN）是一所以本科为主的大学。超过一半的理科学生来自代表性不足的少数群体;许多学生是家中第一个上大学的，经济上处于不利地位。很少有少数民族科学家从事蛋白质生物物理研究;这个项目应该有助于扩大这些数字，从该部门的学生人数不足的大游泳池。该项目还将为CSUN学生提供前所未有的机会，利用现代方法和技术参与前沿研究。对于许多学生来说，这将是他们第一次（也可能是唯一一次）有机会体验真实的研究挑战，并学习故障排除和解决问题的技能。这些新的研究技能将转化为增加自信，改善课程学习，并增加他们将受到启发去攻读生物化学或结构生物学高级学位的可能性。更一般地说，该项目的第三个主要成果将是向更广泛的公众说明，高质量，有影响力的研究可以在主要的本科院校进行。