David Nesbitt of the University of Colorado at Boulder is supported by an award from the Chemical Structure, Dynamics and Mechanism program in the Chemistry division to study state-resolved spectroscopy and dynamics of chemical transients. The overall goals of this experimental program are threefold: i) to exploit high sensitivity direct absorption IR laser methods for study of jet-cooled radicals and fundamental molecular ions in slit supersonic discharges, ii) to augment these spectroscopic tools for quantum state-resolved collision studies at gas-ionic solution interfaces, and iii) to utilize time-resolved fluorescence microscopy methods for study of folding/unfolding kinetics at the single molecule level.
Highly reactive, very-short lived molecular species are important for many processes, ranging from atmospheric chemistry at the marine boundary to chemistry in the interstellar media. Additionally, this project involves using single molecular spectroscopy to study the kinetics of folding and unfolding of RNA molecules which is important to our understanding of many biological processes. A diverse group of individuals are involved the research including women and underrepresented minorities. The PI is also the Director of the "CU Wizards" Scientific Outreach program which organizes hands-on lecture/demonstrations for elementary and middle school students.
Research and Education Activities: The research efforts over the past 3 years of this NSF grant have involved: 1) high resolution IR laser spectroscopy/dynamics of highly reactive molecular and ionic transients and 2) single molecule kinetics of isolated RNA tertiary interactions. NSF support has also been instrumental in providing computational resources and developing jet cooled radical generation methods used to study state-to-state scattering dynamics in crossed supersonic jets and liquid interfaces (supported by AFOSR) and spectroscopy of combustion radicals (supported by DOE). The PI has also been involved extensively with undergraduate, graduate and postdoctoral research training and mentoring. Over the past 3 year granting period, this has resulted in record high productivity levels, with 31 total publications (22 in print, 6 in press, 3 submitted) in premier journals acknowledging both primary and shared support through this NSF program. In addition, results from NSF supported work have been presented in 32 invited lectures at 20 international conferences and 12 university departments and government laboratories. In addition, the PI has served as Director for the last 24 years of the CU Wizards Science Outreach Program, which provides lively and educational lecture demonstrations to 200-300 elementary/middle school children and parents on a monthly basis (10 shows per school year). Integrated over the past 24 years, this has translated into approximately 60,000 "person-hours" of educating young children (and equally often their parents) in the importance of science and scientific research. This program is now so long lived that students who came to the series of shows as lower-schoolers have now grown up and bring their own kids to the program! In terms of achievement of important research results, the last 3 years have been dedicated to a wide variety of chemical physics research areas, which greatly exceeds the word limit allowed. Some highlights include: High resolution spectroscopy in the slit jet spectrometer has continued to provide new challenges and insights into large-amplitude "floppy" dynamics of highly quantum molecular clusters. One remarkable example during the last granting period comes from spectroscopic study of H2 + H2O complexes, which represent critically important collision partners in the interstellar medium and speculated to be relevant in generating population inversions for H2O interstellar maser operation Over the past several years, the Nesbitt group has been actively developing a new and evolving thrust into quantum state resolved collision dynamics at the gas-liquid interface, in particular, based on high resolution direct IR laser absorption and laser induced fluorescence methods. Of particular interest has been the use of open shell radial collision partners such as NO, which are amenable to studies of non-adiabatic electronic state changing collisions in the spin orbit manifold. Over this last granting period, we have explored inelastic collision dynamics at the gas-room temperature ionic liquid (RTIL) interface by scattering of a NO projectile beam, with LIF detection yielding detailed distributions in vibrational, rotational and spin orbit degrees of freedom. Over this last granting period, we have explored novel IR laser based methods for combined spatial and temporal control of aqueous sample temperatures in confocal microscopy. Specifically, we have exploited pulsed near-IR diode-laser light at 1.45 mm to locally heat sub-picoliter "nanocolumns" of water with i) sub-mm footprints, ii) > 104 K/s heating rates and iii) DT = 0-70 K dynamic range via first-overtone excitation in the OH-stretch manifold. Most importantly, access to fast, local, and non-contact heating of subpicoliter volumes creates myriad new opportunities for probing the thermodynamics and kinetics of conformational change at the single-biomolecule level. As a final highlight, Mg2+ is known to be essential for correct folding and catalytic activity in RNA, though the underlying free energy, enthalpy and entropy landscapes of how this promotes formation of a biochemically competent structure has remained elusive. We have exploited temperature-controlled single-molecule FRET (smFRET) microscopy to investigate [Mg2+]-dependent thermodynamics of RNA folding/unfolding for an isolated tertiary interaction. Specifically, we have investigated temperature dependent docking and undocking of a GAAA tetraloop with its 11 nucleotide receptor as function of [Mg2+]. The tetraloop–receptor (TL–R) interaction is a ubiquitous modular motif, making it the subject of intensive kinetic and thermodynamic study. Our work reveals that [Mg2+]-based promotion of the tetraloop–receptor tertiary interaction is controlled by a reduction in the barrier entropy (−TDS‡dock) and the overall entropic penalty (−TDSºdock ) for docking, yet with essentially negligible impact on both the activation enthalpy (DH‡dock) and overall exothermicity (DHºdock). These observations contrast sharply with common expectation that increasing [Mg2+] facilitates folding due to reduced electrostatic repulsion of opposing RNA helices, as this would incorrectly predict a decrease in DH‡dock and DH°dock with [Mg2+]. Instead, we propose that higher [Mg2+] facilitates RNA folding by decreasing the entropic penalty of counterion uptake in the folding transition state and thereby reducing disorder of the unfolded conformational ensemble.
