ИСТИНА

Fluorescent proteins (FPs) are extensively utilized in contemporary immunology as tools for imaging living cells. They facilitate the tracking of FP-labeled molecules, including the interactions of proteins, verification of antigens, and the development of biosensors, among other applications. The available repertoire of FPs is extensively employed for multicolor labeling of cellular structures, allowing for high spatial and temporal resolution in imaging studies. The restricted number of available fluorophore combinations poses a significant challenge for expanding the potential of multicolor labeling techniques. In this study, we present a potential strategy to address this limitation by employing time-resolved detection of fluorescence signals from proteins that differ by a single amino acid at the chromophore center. Recently, we demonstrated the ability of molecular modeling to convert the optical signals of FPs [1]. Utilizing a structure-guided, rational protein design approach for UnaG, a bilirubin-activatable FP derived from Anguilla japonica, we hypothesized that modifications to the hydrogen bonding network surrounding the chromophore would alter the spectral characteristics of UnaG. To confirm this, recombinant mutant UnaG proteins were expressed in the E. coli B834 strain and subsequently purified using immobilized metal affinity chromatography. As a result, wild type and two mutant variants of apoUnaG with modified fluorescence lifetimes were successfully generated. Holoforms were obtained by supplementing the apoforms with human serum albumin-bound bilirubin as the ligand donor. To elucidate the differences among the three holoforms of UnaG, time-resolved fluorescence spectra were obtained following excitation with femtosecond laser pulses at a wavelength of 473 nm. Additionally, genetic constructs encoding the three UnaG variants, each fused to one of the following N-terminal localization signals (LS): nucleoplasmin NLS, ensconsin, or the LifeAct peptide, were co-transfected into the HEK293T cell line using lipofection. Fluorescence lifetime imaging microscopy of the co-transfected cells enabled the distinction of various cellular compartments in a time-resolved, high-resolution manner using the same wavelength (excitation at 473 nm). Thus, our model allows for the use of a single wavelength-excited FP to resolve different cellular structures. A similar system holds significant potential for future immunonogical research. All procedures related to the preparation of protein preparations and spectroscopic studies were supported by the Russian Science Foundation (grant number 23-14-00042). [1] Tsoraev, Georgy V., et al. "Intrinsic tryptophan fluorescence quenching by iodine in non-canonical amino acid reveals alteration of the hydrogen bond network in the photoactive orange carotenoid protein." Biochemical and Biophysical Research Communications 683 (2023): 149119.