Eugene G. Maksimov

In recent years, the eIF4E protein family has been actively studied as one of the major susceptibility factors for Solanaceae plants in relation to potyvirus infections. This makes eIF4E an attractive target for genome editing to generate resistant varieties. The viral protein VPg interacts with eIF4E family proteins. Measuring the in vitro affinity of VPg for different eIF4E isoforms has traditionally been difficult. The main reason for this is the high aggregation propensity of VPg. To overcome this, we used protein engineering to generate a chimeric construct based on a fluorescent protein. This approach greatly facilitated the measurement of affinity between two proteins using surface plasmon resonance (SPR). Our results confirm that the α1–α2 hairpin of PVY VPg is involved in complex formation with eIF4E. These findings provide new insights into the molecular mechanism of this protein–protein interaction.

SignificanceThe noninvasive assessment of oxidative stress in the retinal pigment epithelium (RPE), a key factor in the pathogenesis of age-related macular degeneration (AMD), is an important aspect in ophthalmic diagnostics. Although fundus autofluorescence (FAF) is clinically used, its diagnostic power is limited. Fluorescence lifetime imaging ophthalmoscopy shows promise, but the photophysical underpinnings of lifetime changes in lipofuscin—the dominant RPE fluorophore—remain poorly understood. Lipofuscin granules (LGs) in the RPE are a primary source of fundus autofluorescence and are critically involved in the pathogenesis of AMD. This study characterizes the spectral and lifetime properties of LGs using a fluorescence lifetime imaging microscopy (FLIM) system integrated with a wide-range sensitivity, large-active-area superconducting nanowire single-photon detector (SNSPD) coupled with a standard 50 μm multimode fiber.AimWe present experiments on multispectral fluorescence lifetime imaging with a detailed analysis of the fluorescence decays and spectral profiles of LGs in the RPE cells to probe the molecular oxidative state of RPE lipofuscin, aiming to improve AMD understanding and propose a method for early diagnosis of retinal pathologies.ApproachWe use a cutting-edge FLIM system integrated via a multimode fiber with a large-active-area SNSPD. Unlike previous studies, our approach provides component-resolved and spatially resolved fluorescence decay kinetics across a broad spectral range (500 to 1000 nm), which is typically inaccessible to conventional detectors. LGs were isolated from 100 human (aged 50 to 75 years) donor RPE cells and subjected to in vitro photo-oxidation to simulate AMD-like conditions. Steady-state spectroscopy revealed a blue shift in emission following photo-oxidation.ResultsFLIM analysis, employing a tri-exponential decay model, showed a significant increase in the mean fluorescence lifetime τm from ∼247 to ∼587 ps after photo-oxidation. Crucially, we identified a pronounced direct spectral dependence of fluorescence lifetimes τm versus wavelength: for native LGs, τm increased from 136 to 319 ps at registration range 500 to 650 nm; for photo-oxidized LGs, it increased from 174 to 623 ps over the same region. High-performance liquid chromatography confirmed the degradation of the bisretinoid A2E and accumulation of oxidized derivatives, correlating with the observed photophysical changes.ConclusionsThese findings demonstrate that component-resolved visible-near infrared spectral FLIM provides a sensitive, noninvasive approach to probe the molecular oxidative state of RPE lipofuscin, offering potential for early diagnosis of retinal pathologies.

To introduce noninvasive optical diagnostic methods based on detection of tissue autofluorescence signals into medical practice, it is necessary to correlate the optical response with the functional state of a particular chromophore. Diagnostics of the state of lipofuscin granule chromophores is an important task to assess pathologic changes in the visual system in various diseases, primarily age-related macular degeneration. A key feature of lipofuscin granules (LGs) is the diversity of the chromophores and their susceptibility to oxidation. Here, we used time-resolved emission spectroscopy, confocal fluorescence lifetime imaging microscopy, and high-performance liquid chromatography (HPLC) to follow changes of LG chromophore composition upon photooxidation. In both isolated LGs and LG-loaded retinal pigment epithelium cells (ARPE-19), the distributions of the short lifetime component and its amplitude of LG fluorescence shifted, and the mean lifetime increased upon photooxidation, indicating depletion of population of rapidly relaxing bisretinoids, including A2E, and accumulation of their oxidized species, as confirmed by HPLC data. We applied differential evolution algorithms to decompose LG autofluorescence parameters and identified distinct photooxidation patterns in the presence and absence of antioxidant protection. Delivery of zeaxanthin by water-soluble carotenoprotein ΔNC-AstaP attenuated photooxidation-induced changes in the decay kinetics of both isolated and intracellular LGs, suppressed the accumulation of oxidized bisretinoids, and prevented complete photooxidation. Taken together, our results establish that time-resolved lipofuscin autofluorescence provides a quantitative, label-free readout of chromophore composition and oxidative status that is compatible with functional imaging. We propose that such lifetime-based measurements can be employed for early assessment of retinal pathology and for monitoring antioxidant interventions targeting lipofuscin-induced oxidative stress in emerging clinical techniques such as fluorescence lifetime imaging ophthalmoscopy.

The palette of the fluorogen-activating protein FAST expanded into the far-red region by the development of a novel fluorogen, HBTR-3,5-DOM. This was achieved through a C=O to C=S substitution in the classic hydroxybenzylidene-rhodanine core, which induced a bathochromic shift of over 100 nm. The complexes of HBTR-3,5-DOM with FAST variants pFAST and F62L are characterized by absorption and emission maxima at 640–650 nm and ~670 nm, respectively, and are found to exhibit distinct fluorescence lifetimes. The fluorogen is successfully applied in genetically encoded live-cell imaging together with these FAST variants for various subcellular structures. Furthermore, its potential for multiplexed imaging is demonstrated by the simultaneous discrimination of two targeted proteins using fluorescence lifetime imaging microscopy (FLIM).

Multimodal approaches to diagnostics and therapy have lately attracted considerable attention. A promising combination of fluorescence lifetime and optoacoustic imaging offers complementary advantages: fluorescence signals provide metabolic tissue information, while optoacoustic data assess oxygen status and vascularization. This combination is particularly valuable for the in vivo differentiation of normal and pathological tissues, including cancer. Therefore, a stable platform for testing this equipment is crucial. Indocyanine Green (ICG), a clinically approved near‐infrared fluorescent dye, has limited optoacoustic imaging capabilities in its monomeric form. However, converting ICG into J‐aggregates (IJAs) enhances optoacoustic performance by decreasing fluorescence quantum yield and transferring ICG monomers/dimers to nanoparticles. This study evaluates the optical properties of tissue phantoms containing various ratios of vaterite nanoparticles with ICG and IJA for further use in fluorescence lifetime imaging microscopy (FLIM) and optoacoustic tomography. Loading ICG and IJA into vaterite allows for fine‐tuning of FLIM and OA contrast based on the nanoparticles concentrations. This approach facilitates testing of a novel imaging system combining FLIM and OA modalities, opening up new avenues for preclinical and clinical in vivo diagnostics.

Carotenoids (CAR) are a class of organic pigments that are actively involved in the processes of light absorption and non-photochemical quenching in photosynthetic species. Despite the relatively simple chemical structure, CARs exhibit a specific variety of optical properties, which, in general, can be explained within the framework of the multimode Brownian oscillator model (MBOM). However, being a typical semiclassical theory, MBOM operates with a set of effective parameters necessary to model measured spectra. These parameters can be found only by fitting the experiments and for most organic molecules the determination of these parameters is not always unambiguous. CARs are a good example to apply MBOM, since they have only four active vibrational modes, which strongly affect the lineshape of its absorption spectra. Considering the S₀ → S₂ optically allowed all-trans electronic transition of CARs, we performed the fitting of the lutein and zeaxanthin absorption in different solvents at room temperature. To assess the uniqueness of the quantum models for each CAR, the spectral density function was extended with vibrational modes whose frequencies are overtones of the two main vibrational modes of carotenoids. After evolutionary optimization of the fitting routine the experimental data were fitted with high precision and statistically significant parameters of the model were obtained.

Cyanobacteria use soluble antenna megacomplexes, phycobilisomes (PBSs), to maximize light-harvesting efficiency and small photoswitchable orange carotenoid proteins (OCPs) to down-regulate PBSs in high light. Among known PBS morphologies, the one from the basal cyanobacterial genus Gloeobacter still lacks detailed structural characterization. Here, we reconstructed a cryo–electron microscopy structure of the >10-megadalton Gloeobacter violaceus PBS, with diverging, conformationally mobile bundles of rods composed of stacked phycoerythrin and phycocyanin hexamers, stemming from a pentacylindrical allophycocyanin core belted by auxiliary phycocyanin hexamers. We show how two Gloeobacter -specific multidomain linker proteins, Glr1262 and Glr2806, maintain this bundle-shaped architecture and reveal its differential regulation via nonphotochemical quenching by two OCP types of G. violaceus that recognize separate binding sites within the allophycocyanin core, including lateral cylinders absent in tricylindrical cores.

Fluorogen-activating proteins are powerful molecular tools for microscopy, including functional imaging. These proteins serve as an alternative to GFP-like proteins, as they do not require oxygen for chromophore maturation. However, the restricted selectivity of proteins to chromophores, combined with the limited number of spectral channels of conventional fluorescent microscopes, hinders the development of multicolor synthetic dyes. Additionally, the poor cell and tissue permeability of synthetic chromophores further limits their utility. In this work, we address these challenges by combining time-resolved methods with the rational design of the UnaG protein, which utilizes bilirubin as a natural chromophore. To turn UnaG into a palette of probes for fluorescence lifetime imaging microscopy (FLIM), we solved two practical problems: first, we determined the limits of bilirubin lifetime variations in response to changes in the protein structure and, second, we determined what minimal structural changes can be reliably distinguished by lifetime analysis in cellula. Combining classical point mutagenesis and the translational introduction of noncanonical amino acids, we generated UnaG with fluorescence lifetimes ranging from hundreds of picoseconds to nanoseconds. We explored the potential for further modification of the UnaG protein matrix to optimize spectral and temporal characteristics of bilirubin fluorescence and its quantitative detection through time-resolved approaches.

Pathological processes are often accompanied by alterations of protein conformations in blood serum, making investigation of these structural rearrangements highly relevant for clinical diagnostics. Conformation of albumin, the predominant protein in blood serum, is known to be a sensor of pathologies in the human organism; however, label-free methods for its assessment directly in blood serum samples are lacking. In this work, we present a novel analytical methodology for evaluating albumin conformation using the fluorescence parameters of intrinsic blood serum fluorophores excited in the visible range. We first estimate the contribution of various endogenous fluorophores excited in the vicinity of 400 nm to both steady-state fluorescence and fluorescence decay across picosecond and nanosecond time scales, showing that one of the dominant fluorophores is bilirubin, an albumin ligand. In model experiments, we then demonstrate that the structural and photophysical features of bilirubin make its fluorescence decay at picosecond time scale sensitive to conformation of the protein-bilirubin complex. As a final step, it is demonstrated that changes in the ultrafast fluorescence decay parameters of the bilirubin are sensitive enough to detect biologically relevant differences in albumin conformation in serum across different patients. Specifically, we observed statistically significant differences in blood serum albumin's conformation for patients of different age groups (≤34 years and ≥65 years), suggesting that bilirubin may serve as a promising intrinsic sensor for assessing albumin conformational modifications in blood serum.