CAREER: Conformational Dynamics of SH3 Molecular Recognition

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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.