EJ Fine

All Publications

• Interactive computational and experimental approaches improve the sensitivity of periplasmic binding protein-based nicotine biosensors for measurements in biofluids. bioRxiv : the preprint server for biology Haloi, N., Huang, S., Nichols, A. L., Fine, E. J., Friesenhahn, N. J., Marotta, C. B., Dougherty, D. A., Lindahl, E., Howard, R. J., Mayo, S. L., Lester, H. A. 2024

  Abstract

  We developed fluorescent protein sensors for nicotine with improved sensitivity. For iNicSnFR12 at pH 7.4, the proportionality constant for DeltaF/F0 vs [nicotine] (delta-slope, 2.7 muM-1) is 6.1-fold higher than the previously reported iNicSnFR3a. The activated state of iNicSnFR12 has a fluorescence quantum yield of at least 0.6. We measured similar dose-response relations for the nicotine-induced absorbance increase and fluorescence increase, suggesting that the absorbance increase leads to the fluorescence increase via the previously described nicotine-induced conformational change, the "candle snuffer" mechanism. Molecular dynamics (MD) simulations identified a binding pose for nicotine, previously indeterminate from experimental data. MD simulations also showed that Helix 4 of the periplasmic binding protein (PBP) domain appears tilted in iNicSnFR12 relative to iNicSnFR3a, likely altering allosteric network(s) that link the ligand binding site to the fluorophore. In thermal melt experiments, nicotine stabilized the PBP of the tested iNicSnFR variants. iNicSnFR12 resolved nicotine in diluted mouse and human serum at 100 nM, the peak [nicotine] that occurs during smoking or vaping, and possibly at the decreasing levels during intervals between sessions. NicSnFR12 was also partially activated by unidentified endogenous ligand(s) in biofluids. Improved iNicSnFR12 variants could become the molecular sensors in continuous nicotine monitors for animal and human biofluids.

  View details for DOI 10.1101/2023.01.16.524298

  View details for PubMedID 36712031

• Structural insights into binding of therapeutic channel blockers in NMDA receptors NATURE STRUCTURAL & MOLECULAR BIOLOGY Chou, T., Epstein, M., Michalski, K., Fine, E., Biggin, P. C., Furukawa, H. 2022; 29 (6): 507-+

  Abstract

  Excitatory signaling mediated by N-methyl-D-aspartate receptor (NMDAR) is critical for brain development and function, as well as for neurological diseases and disorders. Channel blockers of NMDARs are of medical interest owing to their potential for treating depression, Alzheimer's disease, and epilepsy. However, precise mechanisms underlying binding and channel blockade have remained limited owing to challenges in obtaining high-resolution structures at the binding site within the transmembrane domains. Here, we monitor the binding of three clinically important channel blockers: phencyclidine, ketamine, and memantine in GluN1-2B NMDARs at local resolutions of 2.5-3.5 Å around the binding site using single-particle electron cryo-microscopy, molecular dynamics simulations, and electrophysiology. The channel blockers form different extents of interactions with the pore-lining residues, which control mostly off-speeds but not on-speeds. Our comparative analyses of the three unique NMDAR channel blockers provide a blueprint for developing therapeutic compounds with minimal side effects.

  View details for DOI 10.1038/s41594-022-00772-0

  View details for Web of Science ID 000802860000002

  View details for PubMedID 35637422

  View details for PubMedCentralID PMC10075384