Chemogenetic Approaches for Interrogation and Influence of Biological Systems

In current biology, there is a noticeable trend in developing new tools and methods for targeting proteins of interest as well as various cell compartments for interrogating their position and function. Fluorogen activating proteins (FAPs) offer an approach for specific protein targeting with a high activation ratio which allows their vast applications. Their inherent modularity allows researchers to modify only fluorogen to alter the properties or function of the resulting fluorogen-FAP complex. This work focuses on a rational design and validation of several new fluorogens for already existing FAP systems.We developed a novel fluorogenic dye, an azetidine modified variant of malachite green esters previously used in our lab, that preferentially labels FAPs located in acidic compartments of the cell due to a shifted carbinol-chromophore equilibrium. This fluorogen-FAP pair shows high colocalization with LysoTracker without noticeable background from protein located in neutral cellular locations. This fluorogen provides a novel chemoselective labeling platform for studies of protein trafficking in acidic compartments of the cell which might be applicable for lysosomal storage disorders.Another fluorogenic dye that we developed is an iodinated analog of the synthetic GFP chromophore that preserves tight binding to a previously published de novo designed protein mFAP2b. In addition to having high activation upon binding to the protein, this fluorogen also demonstrates singlet oxygen production capabilities. Addition of this new fluorogen-fluorophore pair to our toolbox of cellular probes would allow researchers to induce targeted ROS-mediated damage in live cells in two separate genetically targeted locations.In order to make population-scale experiments that include photoablation and targeted protein damage possible, we have worked on the development and validation of several different versions of LED-based illumination setups (Lightbox 1.1, Lightbox 2.0, LightCoffin). With these setups researchers can select what wavelength they want to use for their illumination experiment, if they want to have a flat illumination field or a spread of different light doses received by samples and perform the experiments in a reproducible fashion.</div

Excited-state dynamics of a molecular dyad with two orthogonally-oriented fluorophores

The excited-state dynamics of a T-shaped bichromophoric molecule, consisting of two strong fluorophores, diphenyloxazole and diphenylpyrazoline, directly linked in an orthogonal geometry, was investigated. Despite the weak coupling ensured by this geometry and confirmed by the electronic absorption spectra, this dyad exhibits only weak fluorescence in both apolar and polar solvents, with fluorescence lifetimes ranging from 200 ps in CHX to 10 ps in ACN. Ultrafast spectroscopic measurements reveal that the fluorescence quenching in polar solvents is due to the population of a charge-separated state. In non-polar solvents, this process is energetically not feasible, and a quenching due to an efficient intersystem crossing (ISC) to the triplet manifold is proposed, based on quantum-chemical calculations. This process occurs via the spin-orbit charge-transfer (SOCT) ISC mechanism, which is enabled by the charge-transfer character acquired by the S1 state of the dyad upon structural relaxation and by the orthogonal arrangement of the molecular orbitals involved in the transition. The same mechanism is proposed to explain why the recombination of the charge-separated state is faster in medium than in highly polar solvents, as well as to account for the fast decay of the lowest triplet state to the ground state

Tagging of Endogenous BK Channels with a Fluorogen-Activating Peptide Reveals β4-Mediated Control of Channel Clustering in Cerebellum

BK channels are critical regulators of neuronal activity, controlling firing, neurotransmitter release, cerebellar function, and BK channel mutations have been linked to seizure disorders. Modulation of BK channel gating is well characterized, regulated by accessory subunit interactions, intracellular signaling pathways, and membrane potential. In contrast, the role of intracellular trafficking mechanisms in controlling BK channel function, especially in live cells, has been less studied. Fluorogen-activating peptides (FAPs) are well-suited for trafficking and physiological studies due to the binding of malachite green (MG)-based dyes with sub-nanomolar affinity to the FAP, resulting in bright, photostable, far-red fluorescence. Cell-excluded MG dyes enable the selective tagging of surface protein and tracking through endocytic pathways. We used CRISPR to insert the FAP at the extracellular N-terminus of BKα in the first exon of its native locus, enabling regulation by the native promoter elements and tag incorporation into multiple splice isoforms. Motor coordination was found to be normal; however, BK channel expression seems to be reduced in some locations. Alternate start site selection or post-translational proteolytic processing resulted in incomplete FAP tagging of the BKα proteins in brain tissues. In Purkinje cell somata, FAP revealed BK channel clustering previously only observed by electron microscopy. Measurement of these clusters in β4+/- and β4-/- mice showed that puncta number and cluster fluorescence intensity on the soma are reduced in β4-/- knockout animals. This novel mouse line provides a versatile fluorescent platform for studying endogenous BK channels in living and fixed tissues. Future studies could apply this line to ex vivo neuronal cultures to study live-cell channel trafficking