Biology subjects

Borshchevskiy, V. I.

Publications and source records attributed to Borshchevskiy, V. I..

6 recordsLinked to original sources

bioRxiv2026

Structural insights into human recoverin

Recoverin is a key calcium sensor that controls the desensitization of the visual rhodopsin by GRK1. Previous studies have traditionally been conducted on bovine protein (bRec), while data on human ortholog (hRec) remain scarce. Here, we combine X-ray crystallography, X-ray absorption spectroscopy (XANES), quantum mechanical calculations, molecular dynamics, and functional assays to provide an integrated characterization of hRec. The 2Ca2+-bound hRec structure was solved at 1.60 [A], showing that, unlike bRec, hRec interacts with ROS membranes at physiologically relevant submicromolar Ca2+ levels, due to a species-specific charge distribution that might influence membrane interactions. Both recoverins form a set of Ca2+/Zn2+-bound conformers with improved functional performance. X-ray crystallography (1.85 [A]) and XANES revealed a specific tetrahedral Zn2+ site in 1Ca2+-bound hRec, the first such site reported in the NCS family. In 1Ca2+-bound hRec, zinc promotes the formation of active state, whereas in 2Ca2+-state of bRec, it significantly enhances GRK1 binding, as the latter can complement the Zn2+ coordination. These data refine our understanding of recoverin function in humans and highlight its role as a key link between calcium and zinc signaling in mammalian photoreceptors under normal and pathological conditions.

biochemistry↗

bioRxiv2025

ABEL-FRET bridges the timescale gap in single-molecule measurements of the structural dynamics in the A2A adenosine receptor

The functional complexity of G protein-coupled receptors (GPCRs) arises from their structural dynamics, which span timescales from nanoseconds to minutes. Single-molecule Forster Resonance Energy Transfer (smFRET) enables direct observation of these dynamics in individual diffusing receptors, either freely diffusing in solution, using confocal microscopy, or immobilized on surfaces, using Total Internal Reflection Fluorescence (TIRF) camera-based microscopy. However, these modalities are limited to distinct timescales - faster than milliseconds or slower than hundreds of milliseconds, respectively - due to constraints of observation time and camera frame rate, while the slow dynamics observed in immobilized receptors may be perturbed by tethering. To overcome these limitations, we employed smFRET in combination with Anti-Brownian Electrokinetic (ABEL) trapping to extend the observation window of untethered A2A adenosine receptors (A2AAR) embedded in lipid nanodiscs from milliseconds to seconds. This approach enabled us to characterize the conformational heterogeneity in both apo and ligand-bound A2AAR, effectively bridging the gap between fast dynamics measured in freely diffusing receptors and slow dynamics observed in immobilized receptors. Our results, taken together with previous studies, demonstrate that both inactive-like and active-like conformations are populated in apo and ligand-bound A2AAR and dynamically interconvert. Apo and antagonist-bound receptors are predominantly locked in inactive-like or active-like states, exhibiting only slow exchange dynamics on timescales longer than hundreds of milliseconds. In contrast, agonist-binding not only shifts the receptor population toward the active-like state but also accelerates the conformational exchange by nearly three orders of magnitude: from hundreds of milliseconds to sub-millisecond timescales. These results underscore the dual role of agonists in modulating both the equilibrium and dynamics of receptor conformations. Our study highlights the power of ABEL-FRET to probe structural dynamics of GPCRs, offering valuable insights into GPCR conformational landscapes and aiding the development of more effective GPCR-targeted therapeutics.

biophysics↗

bioRxiv2025

Discovery of excited state proton transfer in flavin-based fluorescent protein with large Stokes shift

Flavin-binding proteins (flavoproteins) are widespread in nature, revealing versatile oxidation-reduction reactions and photochemistry. Flavoproteins derived from LOV domains are used for engineering of ligh-tresponsive tools in optogenetics, as well as fluorescent markers and photogenerators of reactive oxygen species. Despite extensiev efforts, all currently used LOV-derived proteins have similar absorption spectra with maxima around 275, 35-0375, and 450-485 nm. Here, we describe the discovery of a large Stokes shift flavi-nbased fluorescent protein, LSSFbFP, which can be obtainedin vivo and in vitro, with absorption maxima at 340-350 and 395-405 nm. Fluorescence emission of LSSFbFP mirrors that of classical FbFPs with the maximum at ~500 nm. We sho that the protein binds lumichrome as the chromophore and use low temperature and time-resolved spectroscopy, X-ray crystallography and modeling to prove that the apparent Stokes shift of LSSFbFP occurs due to excited state proton phenomena observed in flavoproteni s and pave the way for engineering of new flavin-based molecular instruments.

biochemistry↗

bioRxiv2023

Optogenetic control of lysosome function

Lysosome protective, metabolic, signaling functions are highly dependent on their pH. A lack of tools of high spatial and temporal resolution for pH control is a bottleneck of lysosome related cell research. Light-driven inward proton pump NsXeR, targeted to the lysosomes of mammalian cells, produces lysosome alkalization simply by light. Complementary use of outward proton pumping Arch3 rhodopsins in lysosomes offers an approach to vary pH in a range from around 5 to 6.5 in both directions (de-acidification and acidification). Lyso-NsXeR optogenetics efficiency was demonstrated, in particular, by its ability to inhibit lysosome proteolytic enzymes. Thus, optogenetic monitoring and regulation of the lysosome function, through pH control over a wide range, could serve as an approach to studying fundamental cell processes, and rational drug design.

cell biology↗

bioRxiv2023

Functional GPCR expression in eukaryotic LEXSY system

G protein-coupled receptors (GPCRs) represent an important class of drug targets, and their structural studies facilitate rational drug discovery. However, atomic structures of only about 20% of human GPCRs have been solved to date. Recombinant production of GPCRs for structural studies at a large scale is challenging due to their low expression levels and stability. Here we tested the eukaryotic system LEXSY (Leishmania tarentolae) for GPCR production. We expressed the human A2A adenosine receptor (A2AAR) in LEXSY, purified it, and compared with the same receptor produced in insect cells, which is the most popular expression system for structural studies of GPCRs. The A2AAR purified from both expression systems showed similar purity, stability, ligand-induced conformational changes and structural dynamics, with a remarkably higher protein yield in the case of LEXSY expression.

biophysics↗

bioRxiv2022

The SARS-CoV-2 receptor-binding domain facilitates neutrophil transepithelial migration and nanoparticle uptake in the mice airways

SARS-CoV-2-induced infection is still dangerous. Mouse models are convenient to the investigation of virus-activated immune response mechanisms. However, mice are not proper model organisms to study COVID-19 due to decreased interaction affinity between the SARS-CoV-2 receptor-binding domain (RBD) and mouse angiotensin-converting enzyme 2 (ACE2) compared with human ACE2. In the present study, we propose a mouse model that allows estimating the influence of SARS-CoV-2 on the immune system. To mimic the effects of RBD- ACE2 high-affinity interaction, mice received the ACE2 inhibitor MLN-4760. To simulate virus loading, we applied 100 nm particles suspended in the solution of RBD via the oropharyngeal route to mice. In this model, MLN-4760 application enhanced neutrophil egress from the bone marrow to the bloodstream and RBD attracted neutrophils to the luminal side of the conducting airway epithelium. By contrast, inert 100 nm particles were not potent to stimulate neutrophil recruitment to the conducting airway mucosa. Using this model, and by altering the dosage of the ACE2 inhibitor, nanoparticles, and RBD, one can adapt it to investigate different COVID-19 states characterized with mild or severe airway inflammation. StatementThis study presents a mouse model that allows estimating the influence of SARS-CoV-2 on the immune system and investigates immune cell-model virus particle interactions in the conducting airway mucosa.

immunology↗