Browsing by Author "Drobny, Gary P."
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Item Hydration dependent dynamics in RNA(Journal of biomolecular NMR, 2009) Olsen, Greg L.; Bardaro Jr., Michael F.; Echodu, Dorothy C.; Drobny, Gary P.; Varani, GabrieleThe essential role played by local and collective motions in RNA function has led to a growing interest in the characterization of RNA dynamics. Recent investigations have revealed that even relatively simple RNAs experience complex motions over multiple time scales covering the entire ms–ps motional range. In this work, we use deuterium solid-state NMR to systematically investigate motions in HIV-1 TAR RNA as a function of hydration. We probe dynamics at three uridine residues in different structural environments ranging from helical to completely unrestrained. We observe distinct and substantial changes in 2H solid-state relaxation times and lineshapes at each site as hydration levels increase. By comparing solid-state and solution state 13C relaxation measurements, we establish that ns–ls motions that may be indicative of collective dynamics suddenly arise in the RNA as hydration reaches a critical point coincident with the onset of bulk hydration. Beyond that point, we observe smaller changes in relaxation rates and lineshapes in these highly hydrated solid samples, compared to the dramatic activation of motion occurring at moderate hydration.Item Intermediate Rate Atomic Trajectories of RNA by Solid State NMR Spectroscopy(Journal of the American Chemical Society, 2010) Olsen, Greg L.; Bardaro Jr., Michael F.; Echodu, Dorothy C.; Drobny, Gary P.; Varani, GabrieleMany RNAs undergo large conformational changes in response to the binding of proteins and small molecules. However, when RNA functional dynamics occur in the ns-μs time scale they become invisible to traditional solution NMR relaxation methods. Residual dipolar couplings methods have revealed the presence of extensive ns-μs domain motions in HIV-1 TAR RNA, but this technique lacks information on the rates of motions. We have used solid-state deuterium NMR to quantitatively describe trajectories of key residues in TAR by exploiting the sensitivity of this technique to motions that occur in the ns-μs regime. Deuterium lineshape and relaxation data were used to model motions of residues within the TAR binding interface. The resulting motional models indicate that two functionally essential bases within the single stranded bulge sample both the free and Tat-bound conformations on the microsecond timescale in the complete absence of the protein. Thus, our results strongly support a conformational capture mechanism for recognition: the protein does not induce a new RNA structure, but instead captures an already-populated conformation.Item Solid-State Deuterium NMR Studies Reveal μs-ns Motions in the HIV-1 TAR RNA Recognition Site(Journal of the American Chemical Society, 2008) Olsen, Greg L.; Echodu, Dorothy C.; Shajani, Zahra; Bardaro Jr., Michael F.; Varani, Gabriele; Drobny, Gary P.Solution and solid-state NMR measurements were used together to examine motion in three sites in the HIV-1 TAR RNA. We wished to investigate the dynamics facilitating the conformational rearrangements the TAR RNA must undergo for tat binding, and in particular to characterize the full range of motional timescales accessible to this RNA. Our results demonstrate that the dynamics in TAR involving residues essential to tat binding include not only the faster motions detected by solution relaxation measurements, but also a significant component in the μs-ns timescale.