Biology subjects

Maslov, I.

Publications and source records attributed to Maslov, I..

8 recordsLinked to original sources

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↗

bioRxiv2024

Angiotensin-(1-5) is a Potent Endogenous Angiotensin AT2-Receptor Agonist

BackgroundThe renin-angiotensin system involves many more enzymes, receptors and biologically active peptides than originally thought. With this study, we investigated whether angiotensin-(1-5) [Ang-(1-5)], a 5-amino acid fragment of angiotensin II, has biological activity, and through which receptor it elicits effects. MethodsThe effect of Ang-(1-5) (1{micro}M) on nitric oxide release was measured by DAF-FM staining in human aortic endothelial cells (HAEC), or Chinese Hamster Ovary (CHO) cells stably transfected with the angiotensin AT2-receptor (AT2R) or the receptor Mas. A potential vasodilatory effect of Ang-(1-5) was tested in mouse mesenteric and human renal arteries by wire myography; the effect on blood pressure was evaluated in normotensive C57BL/6 mice by Millar catheter. These experiments were performed in the presence or absence of a range of antagonists or inhibitors or in AT2R-knockout mice. Binding of Ang-(1-5) to the AT2R was confirmed and the preferred conformations determined by in silico docking simulations. The signaling network of Ang-(1-5) was mapped by quantitative phosphoproteomics. ResultsKey findings included: (1) Ang-(1-5) induced activation of eNOS by changes in phosphorylation at Ser1177eNOS and Tyr657eNOS and thereby (2) increased NO release from HAEC and AT2R-transfected CHO cells, but not from Mas-transfected or non-transfected CHO cells. (3) Ang-(1-5) induced relaxation of preconstricted mouse mesenteric and human renal arteries and (4) lowered blood pressure in normotensive mice - effects which were respectively absent in arteries from AT2R-KO or in PD123319-treated mice and which were more potent than effects of the established AT2R-agonist C21. (5) According to in silico modelling, Ang-(1-5) binds to the AT2R in two preferred conformations, one differing substantially from where the first five amino acids within angiotensin II bind to the AT2R. (6) Ang-(1-5) modifies signaling pathways in a protective RAS-typical way and with relevance for endothelial cell physiology and disease. ConclusionsAng-(1-5) is a potent, endogenous AT2R-agonist.

pharmacology and toxicology↗

bioRxiv2023

Monitoring GPCR Conformation with GFP-Inspired Dyes

Solvatochromic compounds have emerged as valuable environment-sensitive probes for biological research, with the chromophore of the green fluorescent protein (GFP) being a well-studied example. In this study, we demonstrate that synthetic analogues of the GFP chromophore can be used to investigate ligand-induced conformational changes in proteins. We synthesized thiol-reactive derivatives of four analogues of the GFP chromophore that exhibit notable solvatochromism. We used these derivatives to label two proteins: the soluble calcium sensor recoverin (Rec) and the transmembrane G protein-coupled A2A adenosine receptor (A2AAR), via cysteines located or introduced in the regions that undergo structural changes upon ligand binding. Two of these dyes showed Ca2+-induced fluorescence changes when attached to Rec. Notably, our best-performing dye, DyeC, when attached to A2AAR, revealed agonist-induced changes in both fluorescence intensity and shape of the emission spectrum. Molecular dynamics (MD) simulations provided mechanistic insights into these changes showing the activation of A2AAR transfers DyeC to a more confined and more hydrophilic environment. Additionally, an allosteric modulator, HMA, induces changes in DyeC fluorescence spectra, indicating a distinct receptor conformation from apo, antagonist, or agonist-bound receptors. Our study demonstrates that GFP-inspired dyes are effective for detecting structural changes in GPCR (G protein-coupled receptors), with advantages such as the ability to perform both intensity-based and ratiometric tracking, red-shifted fluorescence spectra, high extinction coefficient, and sensitivity to allosteric modulation. These dyes expand the toolbox for tracking ligand-induced changes and facilitate new insights into conformational changes induced by allosteric modulators in GPCRs.

biophysics↗

bioRxiv2023

Revealing the Oligomerization of Channelrhodopsin-2 in the Cell Membrane using Photo-Activated Localization Microscopy

Microbial rhodopsins are retinal membrane proteins that found a broad application in optogenetics. The oligomeric state of rhodopsins is important for their functionality and stability. Of particular interest is the oligomeric state in the cellular native membrane environment. Fluorescence microscopy provides powerful tools to determine the oligomeric state of membrane proteins directly in cells. Among these methods is quantitative photoactivated localization microscopy (qPALM) allowing the investigation of molecular organization at the level of single protein clusters. Here, we apply qPALM to investigate the oligomeric state of the first and most used optogenetic tool Channelrhodopsin-2 (ChR2) in the plasma membrane of eukaryotic cells. ChR2 appeared predominantly as a dimer in the cell membrane and did not form higher oligomers. The disulfide bonds between Cys34 and Cys36 of adjacent ChR2 monomers were not required for dimer formation and mutations disrupting these bonds resulted in only partial monomerization of ChR2. The monomeric fraction increased when the total concentration of mutant ChR2 in the membrane was low. The dissociation constant was estimated for this partially monomerized mutant ChR2 as 2.2{+/-}0.9 proteins/m2. Our findings are important for understanding the mechanistic basis of ChR2 activity as well as for improving existing and developing future optogenetic tools.

biophysics↗

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↗

bioRxiv2020

Sub-millisecond conformational dynamics of the A2A adenosine receptor revealed by single-molecule FRET

The complex pharmacology of G-protein-coupled receptors (GPCRs) is defined by their multi-state conformational dynamics. Single-molecule Forster Resonance Energy Transfer (smFRET) is well-suited to quantify dynamics for individual protein molecules, however, its application to GPCRs is challenging; therefore, smFRET has been limited to studies of interreceptor interactions in cellular membranes and receptors in detergent environments. Here, we performed smFRET experiments on functionally active human A2A adenosine receptor (A2AAR) molecules embedded in freely diffusing lipid nanodiscs to study their intramolecular conformational dynamics. We propose a dynamic model of A2AAR activation that involves a slow (>2 ms) exchange between the active-like and inactive-like conformations in both apo and antagonist-bound A2AAR, explaining the receptors constitutive activity. For the agonist-bound A2AAR, we detected faster (390{+/-}80 s) ligand efficacy-dependent dynamics. This work establishes a general smFRET platform for GPCR investigations that can potentially be used for drug screening and/or mechanism-of-action studies.

biophysics↗

bioRxiv2020

Accessing mitochondrial protein import in living cells by protein microinjection

Mitochondrial protein biogenesis relies almost exclusively on the expression of nuclear-encoded polypeptides. The current model postulates that most of these proteins have to be delivered to their final mitochondrial destination after their synthesis in the cytoplasm. However, the knowledge of this process remains limited due to the absence of proper experimental real-time approaches to study mitochondria in their native cellular environment. We developed a gentle microinjection procedure for fluorescent reporter proteins allowing a direct non-invasive study of protein transport in living cells. As a proof of principle, we visualized potential-dependent protein import into mitochondria inside intact cells in real-time. We validated that our approach does not distort mitochondrial morphology and preserves the endogenous expression system as well as mitochondrial protein translocation machinery. We observed that a release of nascent polypeptides chains from actively translating cellular ribosomes by puromycin strongly increased the import rate of the microinjected preprotein. This suggests that a substantial amount of mitochondrial translocase complexes were involved in co-translational protein import of endogenously expressed preproteins. Our protein microinjection method opens new possibilities to study the role of mitochondrial protein import in cell models of various pathological conditions as well as aging processes.

cell biology↗

bioRxiv2020

Metabolic fate of human immunoactive sterols in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) infection is among top ten causes of death worldwide, and the number of drug-resistant strains is increasing. The direct interception of human immune signaling molecules by Mtb remains elusive, limiting drug discovery. Oxysterols and secosteroids regulate both innate and adaptive immune responses. Here we report a functional, structural, and bioinformatics study of Mtb enzymes initiating cholesterol catabolism and demonstrated their interrelation with human immunity. We show that these enzymes metabolize human immune oxysterol messengers. Rv2266, the most potent among them, can also metabolize vitamin D3 (VD3) derivatives. High-resolution structures show common patterns of sterols binding and reveal a site for oxidative attack during catalysis. Finally, we designed a compound that binds and inhibits three studied proteins. The compound shows activity against Mtb H37Rv residing in macrophages. Our findings contribute to molecular understanding of suppression of immunity and suggest that Mtb has its own transformation system resembling the human phase I drug-metabolizing system.Competing Interest StatementA.Gi. and E.S. are employees of MT-Medicals LLC. The other authors declare no competing interests.View Full Text

biochemistry↗