CAREER: Conformational Dynamics of SH3 Molecular Recognition

标题：职业生涯：SH 3分子构象动力学细胞的运作是由蛋白质之间的相互作用协调的。并且理解不同的蛋白质如何能够在复杂和拥挤的细胞环境中识别其适当的结合伴侣是当前重要的科学问题。就像宏观机器一样，蛋白质也有活动的部分。然而，通过实验研究蛋白质运动如何参与识别其他蛋白质是具有挑战性的，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移动。这项研究利用化学生物学和物理化学中最先进的方法，将报告化学键放置在蛋白质结构中的特定位置，以测试蛋白质不同部分的运动如何在结合其伴侣时发生变化。由于蛋白质识别是所有生物学的基础，因此关于什么有助于其保真度的知识对我们的理解以及最终操纵生物系统的能力具有广泛的影响。除了她的研究工作，PI将利用她作为印第安纳州大学妇女科学技术中心女本科生教员导师的角色，结合她与当地女童子军的外联活动，培养同龄人/近同龄人榜样，这些榜样可以帮助发展和提高女性科学概念，并识别这样做的障碍。本研究的目的是评估机制蛋白质动力学通过其有助于分子识别的亲和力和特异性。虽然蛋白质动力学越来越多地调用作为贡献者的蛋白质功能，严格的实验评估其作用已受到挑战的空间异质性的蛋白质和潜在的重要构象状态的快速相互转换。这项研究结合了线性FT和二维红外（IR）光谱的固有高时间分辨率与通过蛋白质工程定点掺入振动报告基团所提供的空间分辨率，以产生酵母Sho 1 Src同源3结构域（SH 3）介导的识别其生理靶点脯氨酸的构象动力学的最高分辨率图像。来源于蛋白Pbs 2的富含肽。许多Pbs 2肽变体和另外的SH 3结构域将用于评价蛋白质和/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，并且特别地测试多特异性由增加的蛋白质和/或蛋白质的量导致的模型。或配体灵活性，其涉及通过构象选择机制在未结合状态下的较慢时间尺度构象动力学和通过构象选择机制在更短时间尺度构象动力学的组合。时间尺度构象动力学通过诱导拟合机制。这些研究应该阐明这些关键的真核生物蛋白质相互作用结构域的分子识别机制与前所未有的生物物理细节。

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.