Site-Specific Infrared Spectroscopy of Conformational Heterogeneity in Proline-Rich Motif Recognition
Megan Thielges
Megan, Indiana University
The recognition of proline-rich (PR) linear sequence motifs by Src homology 3 (SH3) domains mediates a broad range of cellular processes and has become a paradigm system for the study of molecular recognition. Despite extensive study, we lack a complete understanding of the molecular basis of many aspects of the molecular recognition, such the thermodynamics and specificity. Conformational heterogeneity and dynamics have been evoked to develop better functional models, but testing them requires experimental tools with sufficient spatial and temporal resolution to measure different parts of peptides and proteins and capture even rapidly interconverting states that could be involved. We take advantage of the bond-specific resolution and high inherent fast timescale of infrared (IR) spectroscopy, in combination with carbon deuterium labeling with frequency-resolved IR probe groups, to enable local, site-specific characterization of rapidly fluctuating peptide and proteins. We applied this approach to characterize every proline of several PR peptides in the absence and presence of SH3 domains. The IR spectra report on varying extent of conformational heterogeneity along peptides, which appears associated with populations of polyproline and disordered structure. The conformational ensemble at a proline, as well as its change upon binding to SH3 domains, depends on the local sequence context. Continuing experiments seek to uncover whether and how the conformational dynamics contribute to the entropy or specificity of molecular recognition of PR motifs.
Megan Thielges completed her B.S. at Arizona State University (Tempe, AZ) in 2003. She pursued graduate studies in biophysics at The Scripps Research Institute (La Jolla, CA) where she earned a Ph.D. under the direction of Professor Floyd E. Romesberg in 2009. She went on to a postdoctoral fellowship with Professor Michael D. Fayer at Stanford University (Stanford, CA). She joined the faculty at Indiana University in the summer of 2012. Her research focuses on developing and applying linear and multidimensional infrared spectroscopy in combination with chemical and biological methods for site-selective protein labeling to generate residue-specific descriptions of the structural heterogeneity and dynamics of proteins, delineate their contributions to function, and assess the role of dynamics in protein evolution.