CAREER Excited States and their Role in Protein-RNA Binding Dynamics

The recognition and binding of nucleic acids by proteins is a central phenomenon in biology, governing processes that range from the flow of genetic information to viral infection and propagation. Despite the importance, our current understanding fails to capture the conformational complexity and dynamic nature of the recognition and binding process. Molecules like RNA exist as an ensemble of accessible conformations, including rare conformations that are not observable using traditional measurements but can be biologically active. Furthermore, a huge span of timescales can be relevant in the process, from microsecond conformational motions to binding/unbinding dynamics occurring over hundreds of seconds. This study leverages advanced spectroscopy and computational modeling to quantitatively map protein-RNA binding dynamics with unprecedented sensitivity and resolution. It will provide a new perspective in protein-RNA interactions that will advance our fundamental understanding of basic biomolecular processes and bring to light the role of hidden states that mediate biomolecular recognition and binding. The study will also strengthen the training of young biological engineers in skills that are critical in research but rarely part of educational curriculum, such as the design and operation of home-built spectroscopic equipment. The central hypothesis of this study is that protein-RNA binding occurs over a range of time and energy-scales, with some interactions being mediated directly through thermally excited conformations of the target molecule. The recent development of new single-molecule spectroscopic methods, in conjunction with non-perturbative fluorescence labeling using non-canonical nucleotide analogues, has enabled the observation of biomolecular dynamics over a temporal range of microseconds to hundreds of seconds and with a sensitivity of < 0.1% of an ensemble population. The study will investigate the binding of the TAR RNA, a small 30 nucleotide hairpin RNA dominated by local secondary fluctuations, and the stem loop A RNA, a large 80 nucleotide RNA structure with secondary and tertiary fluctuations. Taken together, the two systems provide an elegant exploration of how conformational motions of different time and energy scales contribute to protein-RNA binding. The proposed interdisciplinary approach will provide a quantitative (thermodynamics and kinetics), nucleotide-resolved, and temporally resolved picture of protein-RNA binding. The information-rich experimental data, interpreted with the aid of computational modeling, seeks to build the foundational molecular scale knowledge required to understand and predict protein-RNA interactions. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria. NSF Award ID: 2540408 | Program: 01002627DB NSF RESEARCH & RELATED ACTIVIT,01003031DB NSF RESEARCH & RELATED ACTIVIT | Principal Investigator: John King | Institution: University of New Mexico, ALBUQUERQUE, NM | Award Amount: $600,620 View on NSF Award Search: https://www.nsf.gov/awardsearch/show-award/?AWD_ID=2540408 View on Research.gov: https://www.research.gov/awardapi-service/v1/awards/2540408.html