This NSF MRI award funds the acquisition of a Time Correlated Single Photon Counting (TCSPC) fluorescence lifetime system to expand the research capabilities at Kennesaw State University. The TCSPC instrument allows measurement of excited fluorescent molecules in systems that contain a mixture of species with different lifetimes, and will be used in studies such as cellular signaling, enzyme catalysis and control, and cellular stress response. The instrument would be used by students in research and in courses, enhancing student recruitment into science and providing them training in advanced technologies. The results of the research and teaching efforts will be broadly disseminated through abstracts and peer reviewed publications, as well as by active participation of students and faculty at professional meetings.

Project Report

The instrument, a Time Correlated Single Photon Counting (TCSPC) Fluorescence Lifetime Spectrometer, is capable of operating in several modes. It can take conventional fluorescence spectra in which a molecule excited by light emits longer wavelength radiation by scanning either the wavelength of the exciting radiation from a Xenon light source, or the wavelength of the emitted radiation when exciting at a fixed wavelength, recording in either case the intensity of the fluorescence (emitted light). It can measure fluorescence polarization and fluorescence polarization decay, allowing the rotational freedom of molecules in solution (and hence the size of aggregates) to be estimated. The TCSPC mode, however, uses a pulsed light source, which can be a laser or a powerful LED, typically with a pulse width of 10-50 picoseconds. Fluorescence is maximal immediately after excitation, and decays as the excited molecules return to their ground state, typically on a time scale of a few nanoseconds for good fluorophores. By measuring fluorescence as a function of time, we have been able to detect different molecular conformations in which the fluorescent groups are in different environment. Multiple conformations with different decay times can be detected in the same experiment. We introduced a new dimension into fluorescence work by successfully interpreting changes in fluorescent lifetime in structural terms by considering interactions with other prosthetic groups. Fluorescence detection of FMN environments in Nitric Oxide Synthase Collaborations between three of the PIs who supported the initial application have been successful in using the instrument to learn about conformational changes involved in the mechanism of enzymes and in control of signaling processes. The initial fluorescence experiments revealed a series of obligatory conformations in the nitric oxide synthase catalytic cycle characterized by their fluorescence lifetimes. This work was published in FEBS Journal (Ghosh , D.K., Nahm, N. and Salerno, J.C. (2012) FMN fluorescence in inducible NOS constructs reveals a series of conformational states involved in the reductase catalytic cycle. FEBS J. 2012 279(7):1306-17. Selected for reprint in a special FEBS J virtual issue, Molecular Enzymology) Nitric oxide is produced from the amino acid arginine in a reaction that requires molecular oxygen and electrons from NADPH, the most common source of electrons for biosynthesis, and electron delivery to the catalytic site is the controlled step. We have now collected a great deal of data on the signaling nitric oxide synthase isoforms nNOS and eNOS. Briefly, because of their very different fluorescence lifetimes, we can now distinguish three types of conformations: input conformations in which the cofactor FMN gets electrons from NADPH via the cofactor FAD, output conformations in which FMN donates electrons to a heme cofactor at the site of nitric oxide production, and a series of ‘open’ states in which FMN is not associated with other cofactors. The open state resembles free FMN, with a lifetime of ~4.3 nanoseconds. Interactions with FAD (input state) and heme (output state) reduce the lifetimes to 80 picoseconds and 800 picoseconds in those states. Calmodulin binding speeds up the transitions between these states, leading to changes in the distribution of states within the conformational manifold because not all transitions are equally affected. CaM activation of NOS favors the output and open states at the expense of the majority input state. This provides a paradigm for NOS activation, and in addition gives us a powerful probe to study the effects of other regulators on the enzymes. The initial report of this work appeared in our paper (John C. Salerno, Krishanu Ray, Thomas Poulos, Huiying Li and Dipak. K. Ghosh (2013) Calmodulin activates neuronal nitric oxide synthase by enabling transitions between conformational states FEBS Letters 587(1):44-7). Other papers are in the process of submission and review. The spectrometer has also allowed us to examine the mechanisms of control by phosphorylation, and to observe the effects of mutations that remove or alter control elements. The initial paper in this series, Salerno, J. C., Ghosh, D. K., Razdan, R. E., Helms, K. A., Brown, C. C., McMurry, J. L., Rye, E. A., Chrestensen, C. A. (accepted). Endothelial nitric oxide synthase is regulated in vitro by ERK phosphorylation at S602 Biosciences Report (Open Access journal of the Biochemical Society), shows that phosphorylation of eNOS S602 by the MAP kinase ERK locks the enzyme down in the input conformation. After a delay caused by the opening of a new building that now houses the group, the organic synthesis faculty have begun to use the instrument to characterize their products. The initial publication (Tapu, D., McCarty, Z., Hutchinson, L., Ghattas, C., Chowdhury, M., Salerno, J. C., VanDerveer, D. (2014). A dibenz[a,c]phenazine-supported N-heterocyclic carbene and its rhodium and iridium complexes. Journal of Organometallic Chemistry / Elsevier, 749(1), 134–141) has appeared.

National Science Foundation (NSF)
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Robert Fleischmann
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Kennesaw State University Research and Service Foundation
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