CAREER: Conformational Dynamics of SH3 Molecular Recognition

标题：职业：SH3分子识别的构象动力学细胞的运作是由蛋白质之间的相互作用精心安排的。了解不同的蛋白质如何在复杂拥挤的细胞环境中识别合适的结合伙伴是目前一个重要的科学问题。像宏观机器一样，蛋白质也有活动部件。然而，通过实验研究蛋白质运动如何参与它们对其他蛋白质的识别是具有挑战性的，因为蛋白质是大而复杂的分子，其部分可以在不同的时间尺度上运动。本研究利用化学生物学和物理化学领域最先进的方法，在蛋白质结构的特定位置放置报告化学键，以测试蛋白质在结合其伙伴时不同部分的运动是如何变化的。由于蛋白质识别是所有生物学的基础，了解是什么促成了它的保真度，对我们的理解，并最终对我们操纵生物系统的能力产生了广泛的影响。除了她的研究工作，PI将利用她作为印第安纳大学女性科学技术中心女本科生的教师导师的角色，结合她与当地女童子军的外展活动，培养同龄人/近同龄人的榜样，帮助发展和提高女性的科学观念，并确定在此过程中遇到的障碍。本研究的目的是评估蛋白质动力学对分子识别的亲和力和特异性的作用机制。尽管蛋白质动力学越来越多地被认为是蛋白质功能的贡献者，但对其作用的严格实验评估受到了蛋白质空间异质性和潜在重要构象状态快速相互转换的挑战。本研究结合了线性FT和二维红外（IR）光谱固有的高时间分辨率，以及通过蛋白质工程将振动报告基团的位点选择性结合所提供的空间分辨率，以产生酵母Sho1 Src同源3结构域（SH3）介导的对其生理靶标（源自蛋白质Pbs2的富含脯氨酸的肽）的识别所涉及的构象动力学的最高分辨率图像。许多Pbs2肽变体和额外的SH3结构域将用于评估蛋白质和/或配体构象动力学是否以及如何调节分子识别的亲和力和特异性。特别地，为了验证这个模型，即多特异性是由于蛋白质和/或配体灵活性的增加，包括通过构象选择机制在非结合状态下较慢的时间尺度构象动力学和通过诱导拟合机制的更快的时间尺度构象动力学的组合。这些研究将以前所未有的生物物理细节阐明这些关键真核蛋白相互作用域的分子识别机制。

Title: CAREER: Conformational Dynamics of SH3 Molecular RecognitionThe operation of a cell is orchestrated by interactions among proteins. and understanding how different proteins are able to recognize their appropriate binding partners within the complex and crowded cellular environment is currently an important scientific question. Like macroscopic machines, proteins have moving parts. However experimentally studying how protein motion is involved in their recognition of other proteins is challenging because proteins are large, complex molecules with parts that can move on different timescales. This research takes advantage of state-of-the-art methods in chemical biology and physical chemistry to place reporter chemical bonds at specific locations in a protein structure to test how the motions at different parts of the proteins change upon binding their partners. As protein recognition underlies all of biology, knowledge about what contributes to its fidelity has wide reaching impact to our understanding, and ultimately our ability to manipulate, biological systems. In addition to her research efforts, the PI will utilize her role as a Faculty Fellow mentor for female undergraduates of the Indiana University Women in Science and Technology Center in combination with her outreach activities with the local Girl Scouts to develop peer/near-peer role models who can help develop and enhance female scientific conception and identify obstacles in doing so.The objective of this research is to evaluate the mechanism(s) by which protein dynamics contribute to the affinity and specificity of molecular recognition. Although protein dynamics are increasingly invoked as contributors to protein function, rigorous experimental assessment of their role has been challenged by both the spatial heterogeneity of proteins and the rapid interconversion of potentially important conformational states. This research combines the inherent high temporal resolution of linear FT and 2D infrared (IR) spectroscopy with the spatial resolution afforded by site-selective incorporation of vibrational reporter groups via protein engineering to generate the highest resolution picture possible of the conformational dynamics involved in the yeast Sho1 Src homology 3 domain (SH3)-mediated recognition of its physiological target the proline-rich peptide derived from the protein Pbs2. A number of Pbs2 peptide variants and additional SH3 domains will be used to evaluate whether and how protein and/or ligand conformational dynamics modulate the affinity and specificity of molecular recognition, and specifically to test the model that poly-specificity results from increased protein and/or ligand flexibility involving a combination of slower timescale conformational dynamics in the unbound states via a conformational selection mechanism and faster timescale conformational dynamics via an induced-fit mechanism. The studies should elucidate the mechanisms governing the molecular recognition of these critical eukaryotic protein interaction domains with unprecedented biophysical detail.