CAREER: NMR Insights into the Influence of Dynamics on SH3 Domain Mediated Protein Interactions

该项目的目标是开发新的技术来表征溶液状态下蛋白质的核磁共振（NMR）弛豫。这些技术的发展和实施将促进（i）实验设计和分析的理论方法的进步，（ii）在市售仪器的重大改进，和（iii）开发具有成本效益的方法，用于细菌表达和纯化标记的蛋白质。这些方法将被应用于研究蛋白质动力学在很宽的时间范围内，特别强调蛋白质侧链动力学的表征，因为侧链在定义蛋白质-配体相互作用和生理功能中起着核心作用。这些方法将减轻目前的实验的许多不足之处，（一）提供信息的细节上的快速，ps-ns的时间尺度，（ii）允许完整的表征缓慢，相关的运动上的微秒-ms的时间尺度，（iii）适用于较大的蛋白质和蛋白质复合物。PI将利用分子动力学模拟与理论建模相结合，以确定有助于测量弛豫速率的特定动态模式。这种实验和计算技术的结合将允许识别关键功能模式及其对蛋白质相互作用的变化。PI将应用该方法阐明涉及NADPH氧化酶复合物的两种胞质组分-p47 phox和p67 phox的分子内和分子间相互作用的动态决定因素。SH 3结构域是一个非常重要的结构模块，在这些情况下在定义蛋白质相互作用方面起着核心作用。因此，从这些研究中收集的信息将提供一个自洽的，空间-时间的看法SH 3结构域介导的蛋白质interactions.The PI将特别重视招募学生从不同背景和不同层次的复杂性，本科生和研究生，到他的研究计划。他将重新设计本科课程，为学生提供尖端生物物理技术的知识，并开发一门针对高级本科生和研究生的NMR光谱学新课程。本课程将介绍学生的理论以及现代生物分子核磁共振光谱学的应用方面都通过讲座以及一系列的“动手”研讨会。PI目前参与并将继续参与ACS-SEED等科学推广计划。该计划旨在帮助贫困高中生在夏季获得研究经验。CUNY City College拥有大量的少数民族人口。因此，该项目的研究和教学部分将鼓励那些在科学领域代表性不足的社会阶层在学术界和工业界从事科学事业。通过教学，辅导，职业咨询和参与弱势学生的外展计划促进少数民族参与科学是这个职业项目的一个关键目标。

The objective of this project is to develop novel techniques to characterize Nuclear Magnetic Resonance (NMR) relaxation of proteins in the solution state. The development and implementation of these techniques will be facilitated by (i) advances in theoretical methods for experimental design and analysis, (ii) major improvements in commercially available instrumentation, and (iii) the development of cost-effective methods for the bacterial expression and purification of labeled proteins. These methods will be applied to study protein dynamics over a wide range of timescales, with special emphasis on the characterization of protein sidechain dynamics since sidechains play a central role in defining protein-ligand interactions and hence physiological function. These methods will alleviate many of the deficiencies of current experiments by (i) providing information on the details of local dynamics on the fast, ps-ns timescale, (ii) allowing complete characterization of slow, correlated motions on the microsecond-ms timescale, and (iii) being applicable to larger proteins and protein complexes. The PI will utilize molecular dynamics simulations in conjunction with theoretical modeling to identify specific dynamic modes that contribute to the measured relaxation rates. This combination of experimental and computational techniques will allow the identification of key functional modes and the changes thereof on protein interactions. The PI will apply this methodology to elucidate the dynamic determinants of the intra- and inter-molecular interactions involving two cytosolic components of the NADPH oxidase complex - p47phox and p67phox. A very important structural module, the SH3 domain plays a central role in defining protein interactions in these cases. Thus, information gleaned from these studies will provide a self-consistent, space-time view of SH3 domain-mediated protein interactions.The PI will place special emphasis on recruiting students from various backgrounds and at various levels of sophistication, undergraduate and graduate, into his research program. He will redesign undergraduate courses to provide students with the knowledge of cutting edge biophysical techniques and develop a new course on NMR spectroscopy targeted towards advanced undergraduates and graduate students. This course will introduce students to both the theoretical as well as the applied aspects of modern biomolecular NMR spectroscopy both through lectures as well as a series of "hands-on" workshops. The PI currently participates, and will continue to do so, in science outreach programs such as ACS-SEED. This program is designed to facilitate underprivileged high-school students to gain research experience over the summer. CUNY City College has a large minority population. Thus the research and teaching components of this project will encourage those sections of society underrepresented in the sciences to pursue scientific careers both in academia and in industry. Facilitating minority participation in the sciences through teaching, mentoring, career counseling and participating in outreach programs for disadvantaged students is a key goal of this CAREER project.