Biology

Dr. Sambashiva Banala
Janelia Research Campus, Howard Hughes Medical Institute

Chemical probes offer powerful and precise solutions for many biological problems. Caged probes have their activity turned off but can be activated by light, and such caged probes allow further levels of spatial and temporal control. I designed and synthesized a variety of caged probes for labeling desired proteins via SNAP-tag fusion, a widely used self-labeling system for biological  studies. Specifically, I created caged substrates that require UV illumination to release an active molecule, and a caged fluorophore which becomes fluorescent only after UV illumination. Additionally, details of two novel approaches I recently developed for the synthesis of caged nicotine will be presented. Nicotine is the primary agent in tobacco that causes addiction. It is known that nicotine induces upregulation of nicotinic acetyl choline receptors (nAChRs) but it is unknown whether binding occurs on the cell surface or to nascent proteins in the endoplasmic reticulum. The caged nicotine I developed can be activated with UV illumination at the subcellular level, allowing investigation of the binding of nicotine to nAChRs in the aforementioned locations. The obtained results for nicotine could have further implications in the neuropharmacology of other psychiatric drugs. The approaches I developed are generalizable for caging other molecules which contain a cyclic tertiary amine moiety.

For my future research, I aim to combine chemical probes, especially fluorogenic probes derived from fluorescent proteins, with the SNAP-tag labeling approach. A preliminary study was performed on the fluorophore of green fluorescent protein (GFP). The fluorophore of GFP, 4-hydroxybenzylideneimidazolinone (HBI), is known to be non-fluorescent when chemically synthesized, and its fluorescence in native GFP is due to specific contacts with residues which restrict intramolecular rotation and result in fluorescence. I synthesized a HBI derivative for SNAP-tag labeling and discovered that its fluorescence was recovered upon binding to SNAP-tag. I want to expand this approach to develop fluorogenic probes spanning the visible spectrum. Their biological applications will be investigated in wash free labeling of cells, super-resolution imaging, gap junctional connectivity of neuronal cells, and protein-protein interactions.