Biophysics and Physicobiology

Forster resonance energy transfer (FRET) is a physicochemical phenomenon involving non-radiative energy transfer between donor and acceptor fluorophores. While FRET efficiency primarily depends on the proximity between fluorophores, additional factors also substantially influence the efficiency in living cells. However, how non-distance factors modulate live-cell FRET efficiency remains poorly understood. Here, we report the significant role of N- and C-terminal topology in determining live-cell FRET efficiency, independent of fluorophore proximity, donor variants, and subcellular compartment. Using acceptor photobleaching and sensitized emission measurements in living cells, we found that FRET efficiencies of mCherry-EGFP or mCherry-EYFP (acceptor-donor) were significantly higher than those of EGFP-mCherry or EYFP-mCherry (donor-acceptor), respectively. These efficiencies were comparable between the nucleus and cytoplasm. An orientation index analysis showed that the acceptor-donor configuration is more favorable than the donor-acceptor configuration regardless of donor variants and subcellular localization. FRET efficiencies were also higher with EYFP than with EGFP as the donor. AlphaFold2-based structural modeling suggested similar proximity with structurally heterogeneous and loosely constrained geometry of donor and acceptor fluorophores. Collectively, these results demonstrate that topological arrangement, rather than simple distance considerations, plays a significant role in FRET efficiency in living cells, providing molecular implications for the design of intramolecular FRET-based biosensors.

Stimulated emission depletion (STED) microscopy is a super-resolution fluorescence imaging technique that achieves high spatial and temporal resolution by exploiting stimulated emission to induce fluorescence depletion (FD) and is expected to have substantial utility for imaging applications using fluorescent proteins. However, the compatibility of fluorescent proteins with STED microscopy systems has been understood primarily through empirical observations, and there is no established methodology for the rational selection of fluorescent proteins for STED microscopy. In this study, we systematically evaluated the compatibility of commonly used fluorescent proteins with STED microscopy systems by measuring FD properties using transient absorption spectroscopy and fluorescence dip spectroscopy, both of which are classified as two-color spectroscopy (TCS). Fluorescent proteins identified as compatible with the STED microscopy system based on the TCS measurements were employed for three-dimensional STED imaging of cellular samples expressing each protein. In all samples, three-dimensional spatial resolution was improved relative to confocal laser microscopy, with particularly marked improvements in z-axis resolution. These findings demonstrate that measurements of FD properties via TCS provide a robust approach for evaluating the compatibility of fluorescent proteins with the STED microscopy system and for selecting suitable fluorescent proteins for STED imaging.

Invertebrate vision relies on bistable visual pigments flipping upon photon absorption between rhodopsin and metarhodopsin states. In living butterflies, the UV-VIS absorption spectra of rhodopsin and metarhodopsin, respectively with 11-cis and all-trans isomers of 3-hydroxy-retinal (A3) chromophore, can be conveniently recorded from the eyeshine, the light reflected from the compound eye after passing twice through the light-guiding rhabdoms. * Here, a microscope coupled with a broadband LED source and a microspectrometer was used to record photorelaxations reported in eyeshine reflection spectra. Fitting temporal exponential relaxations to log-reflectance arrays yielded transient and baseline spectra that are analogous to absorbance difference and sum, respectively. Both types of spectra were subjected to singular value decomposition and to fitting of templated visual pigment absorption spectra. * The compound eye of the high brown fritillary Fabriciana adippe was exposed to a series of second-long broadband light pulses, causing photorelaxations with time constants between 40 and 120 ms that led to 80% metarhodopsin in equilibrium. The transient and baseline spectra were fitted with pigment templates, estimating the alpha peak wavelength 547-552 nm for rhodopsin and 496-501 nm for metarhodopsin. The metarhodopsin to rhodopsin alpha peak absorbance ratio 1.25-1.35 is consistent with the isosbestic wavelength at 530 nm. The second isosbestic wavelength indicates that rhodopsin beta (UV) peak absorbs more strongly than metarhodopsin below 405 nm. * Baseline spectra, which were not explicitly analysed in previous studies, enable concatenation of exposures, monitor long-term changes of pigment, and enhance the estimation of beta peak parameters. * The method can be directly used in many butterflies and could be adapted to other insects, particularly fruitflies, facilitating studies of the relation between the visual pigment spectra and the opsin sequences. Spectroscopic results can be complemented with physiologically measured photoreceptor spectral sensitivity datasets and analysed with the same global fitting procedure.

Photoactivated intermolecular electron transfer (ET) in cryptochromes proceeds along chains of aromatic residues and creates a spatially separated pair of radical electrons. Ultrafast time-dependent spectroscopy can provide experimental insight into this process and theoretical estimates of charge transfer rates are commonly obtained via Marcus theory. Here, we present a new perspective on the ET in European robin cryptochrome 4a (ErCry4a) that synthesizes insights from real-time ET calculations, ultrafast spectroscopic measurements and analytical derivations. The simulations exemplify that molecular vibrations play an essential role in enabling the ET dynamics, which was further rationalized through analytical derivations. Ultrafast pump-probe spectroscopy provided experimental access to the first 1.5 ns of the ET cascade, where multiple radical pair recombination rates arise due to the dynamic equilibrium along the ET chain. We show that the motions of the protein environment and the ET dynamics are inseparably coupled, violating the timescale separation required for Marcus theory. The presented results highlight that non-equilibrium coupling between electronic and nuclear motion dominates ET kinetics in ErCry4a during the first nanosecond after photo-excitation. The findings exemplify the limits of Marcus theory and refine the interpretation of ultrafast spectroscopic signatures in cryptochromes.

Fluorescence lifetime imaging microscopy (FLIM) of endogenous NAD(P)H enables the label-free assessment of cellular metabolic state. Although metabolic imaging is increasingly combined with fluorescent protein (FP) reporters to enhance biological specificity, the potential cross-talk between the intrinsic and extrinsic labels remain ill-defined. Here, we systematically evaluate cross-talk from FPs in metabolic FLIM using phasor analysis of two-photon fluorescence microscopy. The results clearly show that many widely used fluorescent proteins are excited under the conditions used for NADH imaging; they emit blue-shifted, short-lifetime fluorescence that can interfere with imaging NADH metabolic signatures. This overlap persists across excitation wavelengths and FP classes, posing a significant challenge for multiplexed metabolic imaging. This cautionary tale argues against unvalidated multiplexing strategies in metabolic FLIM studies. Our study aims to identify acceptable imaging partners, offer a pipeline for assaying potential cross-talk, and provide practical guidance for experimental design. SignificanceFluorescence lifetime imaging of NADH autofluorescence is a powerful, label-free approach to map cellular metabolism in living tissues. A growing number of studies combine NADH imaging with fluorescent protein (FP) reporters to simultaneously identify specific cell types or subcellular compartments. This study reveals that many FPs, spanning the visible spectrum, are unexpectedly excited under NADH conditions. Commonly used green, yellow, and red variants produce short-lifetime, blue-shifted fluorescence that directly overlaps with metabolic NADH signals. This cross-excitation can be falsely interpreted as a shift in cellular metabolic state, posing a significant risk for multiplexed metabolic imaging studies. Our studies establish a pipeline to assess and manage this risk. We identify StayGoldE138D and mNeonGreen as the most compatible FPs for co-imaging with NADH, and provide a practical framework to guide experimental design and control strategies for multiplexed metabolic FLIM.

Fluorescence imaging of dense cellular regions of interest (ROIs) in cells using fluorescence microscopy provides detailed images with pixels that report ensemble- and time-averaged biomolecular data, due to the diffraction limit when super-resolution modalities are not used and acquisition times are slower than typical biomolecular mobilities. The fluorescently-tagged biomolecules that are undergoing imaging can be more heterogeneous and dynamic, all within the dimensions of a single acquired image pixel. The ability to acquire data one biomolecule at a time within a given ROI can help recover some of the underlying biomolecular subpopulations that are otherwise averaged out. In this work, we present a relatively simple approach to achieving single-biomolecule photon bursts, BLeaching In-cell Single-molecule burstS (BLISS). We reveal millisecond photon bursts arising from clusters of mCherry-tagged heterochromatin protein 1 (mCherry-HP1) within heterochromatin biomolecular condensates in undifferentiated mouse embryonic stem cells (ESCs). Fluorescence lifetimes of these bursts are substantially lower than the averaged-out values observed per pixel in fluorescence lifetime imaging microscopy (FLIM), attributed to higher density in mCherry-HP1 clusters. These higher density clusters are observed primarily in undifferentiated ESCs. Two days after retinoic acid (RA) induction of differentiation, these bursts are rarely observed. In summary, using BLISS, we revealed a rare subpopulation of dense mCherry-HP1 clusters characterized by rapid, millisecond dynamics. These clusters are part of heterochromatin biomolecular condensates in ESCs at the pluripotent state, which would be otherwise averaged out in diffraction-limited fluorescence microscopy.

Fluorescent voltage-sensitive dyes (VSDs) enable non-invasive, high-throughput optical measurement of membrane potential in living cells, but the analytical reliability of such measurements depends critically on whether the dye and associated imaging conditions perturb the system under study. Here, we systematically characterise the photophysical performance and cell-perturbing effects of FluoVolt, a widely adopted VSD, across cancer cell lines (GIN31 glioblastoma and SK-MEL-30 melanoma) and primary human macrophages. Photobleaching kinetics were strongly cell-type-dependent, with SK-MEL-30 cells exhibiting complete fluorescence loss within 400 seconds under standard widefield conditions. FluoVolt staining combined with laser excitation caused an approximately 2.5-fold increase in cell detachment relative to unstained controls, and dual-wavelength excitation (488 + 405 nm) reduced GIN31 cell viability by approximately 17.5%. Critically, morphological changes, a transition from elongated to amoeboid-like phenotypes, were detected under staining conditions alone, prior to any laser exposure, indicating baseline dye-induced perturbation independent of phototoxicity. Halving dye concentration and loading time significantly attenuated these effects while preserving measurable fluorescence signal. These findings identify FluoVolt staining and excitation as previously uncharacterised sources of systematic measurement artefact and provide practical, actionable guidance for protocol design, control selection, and data interpretation in optical membrane potential imaging.

Temperature is a fundamental parameter governing all molecular processes, including those that define life. Fluorescence microscopy is a powerful tool to observe molecular processes in living systems in real time. Precise control and measurement of temperature during fluorescence microscopy is therefore essential. We present here a robust temperature measurement based on the excited-state lifetime of the widely available and relatively inexpensive fluorescent dye pentamethine cyanine (Cy5). The excited-state lifetime of Cy5 shows a monotonic decline in the measurement range of 0 {degrees}C - 80 {degrees}C. The measured dependency is linear until 39 {degrees}C and monoexponential above. The dependance of excited-state lifetime upon temperature is used to measure temperature up to a precision of 0.5 {degrees}C or less, a temporal resolution down to <1 millisecond and to resolve temperature gradients with spatial resolutions that are only diffraction-limited. The far-red excitation and emission of Cy5 leaves bandwidth to simultaneously measure at least 3 additional spectral channels in standard fluorescent microscopes simultaneously. We demonstrate determination of temperature during 4-color live-cell fluorescence microscopy for a temperature-controlled experiment. We also show its applicability in measuring temperature gradients and laser-induced sample heating such as during STED nanoscopy.

Magnetic treatment in tomato seeds (Solanum lycopersicum L.) has been studied as a biotechnological technique to induce a reduction in germination times and enhance plant development. However, the modified cellular mechanisms involved in the reduction of germination times or the improvement of development parameters are not yet clearly established. To explore a possible altered cellular mechanism, the effect of homogeneous static magnetic fields on the structure of the cyclic nucleotide-gated channel 6 (CNGC6), the modification in the organization of POPC lipids in the plasma membrane, and changes in calcium ion mobility were evaluated. For this purpose, coarse-grained molecular dynamics simulations were performed using the Martini 3 model in GROMACS, applying five different magnetic flux densities (0.000, 0.001, 0.010, 0.100, 1.000, and 10.000) T over 1 000 ns. The results showed an anisotropic effect in the longitudinal direction of the protein, which generated heterogeneous behavior among the chains of the homotetramer; this altered the conformation of the CNGC6 channel and modified the pore bottleneck. In contrast, no significant changes were observed in the conformational order of the POPC phospholipid chains. As a preliminary, single-replicate exploratory study, these results suggest that homogeneous static magnetic fields may induce specific structural modifications in the CNGC6 ion channel of Solanum lycopersicum L. without compromising the integrity of the lipid bilayer or the dynamics of ion transport within the analyzed timescale; these preliminary findings provide a molecular-level structural basis for future experimental and computational investigations of magnetic field effects on plant cyclic nucleotide-gated channels.

When three cyanobacterial proteins--KaiA, KaiB, and KaiC--are incubated with ATP in vitro, the phosphorylation level of KaiC exhibits stable circadian oscillations. Biochemical and structural analyses have shown that KaiCs ATPase activity is crucial for these oscillations, leading to the hypothesis that ATP-consuming dynamics function as a molecular clock, determining the oscillation period of individual molecules. Moreover, these molecular clocks synchronize with one another, resulting in collective oscillations at the ensemble level. In this study, we develop a theoretical model to test this molecular clockwork hypothesis. Our model clarifies the relationship between the oscillation period and ATPase activity, explaining the significant changes in the period induced by amino-acid substitutions near the CI-CII domain boundary of the KaiC hexamer. Furthermore, the model addresses the physical basis for temperature compensation concerning both the oscillation period and ATPase activity. Thus, the molecular clockwork perspective provides a framework for understanding the atomic design behind collective oscillations.

We demonstrate the use of fluorescence correlation spectroscopy (FCS) to characterize fluorescently-labeled protein production. We use cell-free protein synthesis to express the protein YFP-CopB, a fusion of Chlamydia Outer Protein (Cop) B and Yellow Fluorescent Protein (YFP). CopB is a [~]50 kDa protein believed to have a critical role in chlamydial infection.1 After adding a plasmid encoding YFP-CopB to an E. coli cell-free lysate, protein expression begins. We track the cell-free reaction over several hours using the EI-FLEX, a commercial instrument with FCS capability. As protein is expressed over time, YFP-CopB increases in concentration, and the EI-FLEX detects an increase in fluorescent signal above the background of the cell-free lysate. The FCS data collected gives information about the size, aggregation tendencies, rates of production and fluorescent protein maturation, and concentration of the YFP-CopB produced. The use of FCS concurrent with cell-free synthesis presents a simple method to characterize proteins of interest as they are produced without the need for purification.

Generation of otic progenitors from pluripotent stem cells requires precise timed regulation of signalling pathways, including bone morphogenetic protein 4 (BMP4). Because endogenous levels of BMP4 varie between cell lines, the optimal concentration of exogenous BMP4 must be determined individually to achieve efficient otic differentiation. Three different human induced pluripotent stem cell lines (hiPSCs) underwent ectodermal differentiation to early otic induction stages in the presence of various concentrations of BMP4 (0-5 ng/ml). Differentiation outcomes were assessed by immunofluorescence staining, and quantitative gene expression analysis. Raman microscopy was used to characterize biochemical differences between hiPSC differentiated cultures exposed to different BMP4 concentration. We observed distinct ectodermal fate were after 8 days of in vitro differentiation depending on BMP4 concentration, including neural, non-neural/otic ectoderm and surface epidermal fates. The proportion of PAX2-otic progenitors varied substantially between cell lines and culture conditions, ranging from approximately 9% to 77%. Raman spectroscopy revealed concentration dependent spectral differences and enabled discrimination between differentiating condition within individual hiPSC lines. Analysis of Raman spectral features indicated differences in nucleic acid, lipid, protein, and collagen associated signatures across culture conditions and cell lines. These findings demonstrate that Raman microscopy provides a non-destructive, label-free method for monitoring molecular changes associated with early otic differentiation. By complementing conventional molecular and immunocytochemical analyses, Raman spectroscopy offers a valuable tool for optimizing BMP4-mediated otic induction protocols and improving the reproducibility of stem cell-based strategies for inner ear research and regenerative medicine.

Imaging the structural organization inside cells in their native state is essential for understanding how the arrangement and interactions among molecular components give rise to biological function. Fluorescence microscopy is one of the pivotal techniques that provides molecular specificity for imaging in real space, however, the technique is limited to labeled components. X-rays, on the contrary, are sensitive to electron density contrast and therefore to label-free samples, and probe structure in reciprocal space. In particular, scanning small-angle X-ray scattering (SAXS) combines information from real and reciprocal space and enables access to intact cells, owing to the high penetration power of the X-rays. Combining both imaging modalities in a synergistic manner promises powerful tools for cellular imaging, but remains challenging, because of the differing requirements the complementary methods introduce. Here we present a correlative imaging platform that integrates a modular, compact and beamline-compatible fluorescence microscope with scanning SAXS, to enable fast sequential imaging of the identical cellular regions. We developed a dedicated microfluidics flow chamber enabling measurements under hydrated, near-native conditions. We demonstrate the utility of our methodology by investigating two different relevant cellular components, i.e., thick keratin bundles in epithelial cells that contribute to cell mechanics, and force-generating actomyosin in cardiomyocytes. Employing adapted data analysis methods, we find a good agreement between the fluorescence-derived and the SAXS-derived orientation maps. This result demonstrates that the label-free approach with SAXS captures cytoskeletal organization through-out the cell, and can be directly linked to specific molecular information provided by the complementary fluorescence imaging, in a physiologically relevant cellular environment. Our work establishes a general strategy for multimodal imaging of cellular architecture and opens ways to investigate living cells under the influence of drugs and chemical manipulation experiments.

1.We report a repetition-controllable gain-managed nonlinear fiber amplifier (GMNA) that delivers near-infrared 50-fs pulses with pulse energies up to 150 nJ and a widely tunable repetition rate from 1-20 MHz, while maintaining stable pulse quality across the full range. Using this source, we demonstrate label-free multiphoton imaging--including metabolic autofluorescence (2PF/3PF), second/third-harmonic generation, and Simultaneous Label-free Autofluorescence Multiharmonic (SLAM) microscopy imaging--across live cells, human lung spheroids, and hard tissues. We further assess the impact of laser repetition rate on photodamage at fixed pulse energy, supported by preliminary measurements indicating lower damage at lower repetition rate. Collectively, the compact architecture and repetition-rate agility of the GMNA enable real-time optimization of imaging speed, depth, and sample safety for advanced biological microscopy.

SYBR Gold is one of the most strongly fluorescent members of the SYBR family of asymmetrical cyanine dyes commonly used for staining double and single-stranded DNA and RNA. The dye fluorescence increases dramatically upon nucleic acid binding. The primary mode of binding to double-stranded DNA is through intercalation between the base-paired deoxyribonucleotides, while the precise mode of binding to single-stranded DNA and to RNA is less well understood. Here we show that the fluorescence activity of SYBR Gold is strongly influenced by the presence of guanines in single-stranded nucleic acids. We expect this insight to aid in the evaluation of SYBR Gold-based measurements of nucleic acid abundance and spark further studies into the binding behaviour of the SYBR family dyes.

Mechanotransduction via the actin cytoskeleton is linked to fundamental cellular processes such as morphogenesis, cell division, and motility, requiring the control of tensile forces mediated by the motor protein non-muscle myosin 2 (NM2). Formins such as mDia1 have been shown to elongate actin structures that are under mechanical tension; conversely, mDia1s elongation rates are modulated by the applied force. Despite their relevance at the membrane/cortex interface, reported values for tension in formin-elongated actin filaments stem from theoretical estimates and simulations, but have not been amenable experimentally so far. Thus, we developed a Forster resonance energy transfer (FRET)-based, tension-sensitive probe (mDia1TS) and quantified the measured tension in live U2OS cells using fluorescence lifetime imaging microscopy (FLIM). Through whole-cell ROI analysis we show a short and long lifetime component, reporting an intensity-weighted, averaged lifetime corresponding to [~]3.5 pN. Upon mitogen stimulation of cells using EGF, we show that the tension homeostasis changed significantly, with a measurable increase in tension in the cells periphery and relaxation in its center. Furthermore, the reported average tension relaxed by 2 pN after adding the NM2 inhibitor para-nitroblebbistatin. We utilized siRNA knockdowns of individual NM2 paralogs (NM2-A, NM2-B, or NM2-C) to measure their individual contribution, revealing NM2-A as the main paralog to produce tensile force in this system. Taken together, we demonstrate that mDia1TS is able to directly determine that active mDia1 in cells is under tension, and that subcellular quantification with pN precision is possible. SignificanceDespite the fundamental importance of formins in regulating actin-based processes, reported values for tension in formin-mediated actin structures stem from simulations and theoretical estimates. In this study we developed a FRET-based, tension-sensitive reporter probe for formin mDia1, which we termed mDia1TS. Given the expanding clinical spectrum of DIAPH1/mDia1 mutations, our tool mDia1TS provides a quantitative tool for elucidation of changes in cytoskeletal assemblies.

Systems biology aims to develop predictive models that connect molecular mechanisms to cellular behavior. Genome-scale metabolic models are among the most widely used frameworks for integrating stoichiometric, thermodynamic, and omics-derived information to predict feasible metabolic phenotypes. However, cellular metabolism operates on timescales governed by enzyme kinetics and by the relationship between metabolic fluxes and metabolite pool sizes. In steady-state metabolic models, this relationship can be expressed in terms of metabolite turnover rates, defined as flux-to-pool-size ratios that quantify how rapidly metabolite pools are renewed. As a result, physiologically consistent steady-state solutions should not only satisfy mass-balance and thermodynamic constraints but also exhibit turnover rates consistent with enzyme-mediated cellular dynamics. Current constraint-based approaches can admit many steady-state flux-concentration states that do not account for turnover rates, resulting in phenotypes incompatible with realistic metabolic dynamics, even when multiple types of data are imposed. Here, we present METEOR-K, an optimization framework that links steady-state metabolic fluxes to metabolite concentrations via turnover rate constraints to identify dynamically plausible flux-concentration reference states. Because these constraints reshape the feasible solution space, we also introduce turnover-rate-aware sampling strategies to efficiently explore the resulting feasible region. We applied METEOR-K to models of increasing scope and scale, including a reduced glycolysis pathway, anaerobic E. coli, and near-genome-scale ovarian cancer models. METEOR-K narrowed the admissible steady-state solution space, reduced uncertainty in feasible flux-concentration states, and improved local dynamic behavior. In nonlinear ODE simulations of bioreactor cultivation and drug-response scenarios, METEOR-K-derived states produced intracellular response times compatible with growth-supporting metabolic operation and perturbation recovery. Overall, these results establish metabolite turnover rates as scalable biophysical constraints that improve the physiological consistency of steady-state metabolic modeling. Because turnover rates encode flux-to-pool-size timescale constraints, METEOR-K moves part of physiological-consistency assessment upstream of kinetic parameterization, yielding better-suited flux-concentration reference states for kinetic modeling and dynamic prediction.

The repair of photo-induced DNA lesions through nucleotide excision repair machinery is still the source of important questions. It has been observed that the repair rate of the different cyclobutane pyrimidine dimers, i.e. the photoproducts induced by dimerization of two {pi}-stacked pyrimidines (T<>T, T<>C, C<>T, C<>C), depends on the nucleobases involved in the lesion. TT derivatives (T<>T) are removed more slowly than those containing cytosine, especially in 5. Using all-atom molecular dynamics simulations and free-energy calculations, we demonstrate that the variation of the repair rate observed in human skin and in cultured cutaneous cell is associated to the recognition of the four lesions by the DDB2 protein moiety, and more specifically by the differential structural deformation induced on the complementary strand. Indeed, while C<>C and C<>T induce a larger deviation on the groove parameters, T<>T and T<>C, instead, affect DNA structure to a lesser extent. less affected. These effects then hamper differentially the downstream recruitment of the repair complexes. The observed DNA deformation correlates with the experimental repair rate and provides a structural rationale for the different repair rates of CPD by nucleotide excision repair machinery. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/724087v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@cf6b6dorg.highwire.dtl.DTLVardef@195e35forg.highwire.dtl.DTLVardef@1829296org.highwire.dtl.DTLVardef@165baba_HPS_FORMAT_FIGEXP M_FIG C_FIG

Weak magnetic fields affect many biological processes across the tree of life, though the precise molecular sensors and pathways involved in such magnetoresponses remain mostly uncharacterized. Fluorescence is a useful tool for investigating magnetic field effects in flavoproteins, as their chromophores fluorescence intensity can be shown to depend on the spin states of electronic radical pairs. Here, we describe a four-state ordinary differential equation model to understand what parameter sets result in fluorescence contrast between spin states in photocycles with singlet and triplet radical pairs. We conclude that only certain sets of parameters result in the fluorescence intensity being a good proxy measurement for singlet yield. In particular, we observe that the illumination intensity required to obtain fluorescence contrast depends on the rate of the slow spin-independent radical termination reactions that recover ground-state oxidized fluorophores. Moreover, to observe a magnetic field effect in fluorescence intensity when an external magnetic field modulates the singlet yield, the illumination intensity must be strong enough such that photoexcitation is not the rate-limiting step. This understanding suggests that flavoproteins that do not exhibit magnetic field effects in their fluorescence emission under certain experimental setups may still be sensitive to weak magnetic fields in terms of function, as magnetosensitivity in fluorescence depends strongly on illumination conditions.

A priori assessment of target proteins' druggability remains an unsolved problem in the field of drug development. The empirical approaches widely used to solve this problem demonstrate low efficiency. In this work, we investigated the factor of hydration of a representative set of 65 evolutionarily and structurally unrelated human enzymes in a water environment. This factor depends only on the structure of the proteins, and not on the physical and chemical properties of any potential ligands. The results show that, unlike the widely used approaches based on calculations of the accessible surface area (ASA), the content of low-entropy water molecules (LEW) in the active sites of human enzymes is systematically higher than that in other areas of their surface, including inactive cavities. Optimal criteria and a step-by-step procedure for identifying protein ligand binding sites are proposed. The proposed approach, based on the calculation of the LEW content in the first hydration layer of potentially interesting target proteins, makes it possible to evaluate their medicinal suitability even before the development of any ligands. The article also presents the results of a comparative analysis of experimental Raman spectroscopy data and the results of molecular dynamics simulations of water hydrogen bonds using three widely used water models (TIP3P, OPC3, and TIP5P) and standard algorithms for calculating hydrogen bond networks.