Project Summary RNA chaperone proteins act as versatile matchmakers, delivering regulatory RNAs to their targets to rapidly modulate gene expression in response to environmental signals. In bacteria, the Hfq chaperone flexibly matches small non-coding RNAs (sRNAs) to complementary sites in target mRNAs to regulate translation and turnover of transcripts crucial for bacterial stress response, persistence, and virulence. Previous studies have offered insight into how Hfq mediates the sRNA-mRNA annealing process and the crucial roles of arginine residues on the lateral rim of the Hfq hexamer and intrinsically disordered C-terminal domains (CTD) that gate RNA binding and release from Hfq. These findings have provided a foundational understanding of Hfq’s chaperone activity, but do not address how this chaperone activity is achieved. The goal of this project is to develop a comprehensive, atomistic model describing RNA binding and diffusion on the Hfq chaperone. Aim 1: To use confocal single molecule Förester resonance energy transfer (smFRET) to describe how sRNAs diffuse on Hfq, and to decipher the role of the CTDs in sRNA diffusion. Aim 2: To use 19F nuclear magnetic resonance (NMR) spectroscopy to characterize how Hfq and sRNA dynamics are mutually affected during complex formation. Aim 3: To use all-atom molecular dynamics (MD) simulations to gain atomistic insight into changes in local Hfq and RNA dynamics that enable diffusion and annealing. Collectively, these results will offer a mechanistic understanding of fast dynamics that give rise to Hfq’s RNA chaperone activity. This mechanistic understanding will be applicable to many other RNA chaperones and RNA-binding proteins and will inform the development of next-generation antimicrobials that target bacterial virulence. Carrying out this research will deepen my knowledge of computational simulations while providing me with new skills in two biophysical experimental methods, confocal smFRET and NMR. My training will be conducted as part of the Ph.D. Program in Molecular Biophysics at Johns Hopkins University, which provides rigorous scientific development and monitoring of students. A mentoring committee with expertise in all of the research aims will meet with me regularly to provide scientific and professional advice, and to monitor my training and research progress. I will have access to world-class instrumentation and all necessary resources for completing each specific aim. Additionally, the collaborative and interactive Johns Hopkins biophysics community will offer abundant opportunities for scientific communication, networking and career development.