Biology subjects

Hendrix, J.

Publications and source records attributed to Hendrix, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Social anchors: how previous social locations shape current animal movement and habitat selection

Memory of past space use is an important factor in habitat selection, but memory of what? The concept of memory captures the geographic spaces that an animal has used in the past. There are, however, competing reasons why animals occupy a given space. Species may occupy space due to particular aspects of their memory. Using integrated step selection analysis, we tested the relative importance of memory: previous use of high forage quality or where social interactions occurred at two temporal scales in caribou (Ranger tarandus). The most important parameter to explain intra-seasonal movement was geographic memory. At the inter-annual scale, caribou more strongly revisited locations with high quality forage and where they had formed dyads with conspecifics. We posit that remembered locations of social fusion can function as social anchors. The effects of memory on movement behaviour depend upon the context, both social and resource-based, and not geographic space alone.

ecology↗

bioRxiv2026

Adaptive immunity shapes baseline physiology of M. tuberculosis in high-dose versus low-dose infection BALB/c mouse drug treatment models

Preclinical tuberculosis (TB) drug evaluation relies heavily on mouse infection models. Drug efficacy varies depending on inoculum and the timing of treatment initiation. These differences reflect, in part, physiological adaptations of Mycobacterium tuberculosis (Mtb) to host immune pressure. We used novel molecular markers of pathogen health (RS ratio and SEARCH-TB) to contrast Mtb phenotypes in the BALB/c high dose aerosol (HDA) subacute TB and low dose aerosol (LDA) chronic TB infection models, focusing on the transition from innate to adaptive immunity. We found that the onset of adaptive immunity coincided with a rapid reprogramming of Mtb physiology, characterized by suppressed respiration, metabolism, and biosynthesis, together with induction of stress and nutrient acquisition pathways. Changes in key Mtb processes were concordant with changes in host expression of canonical features of adaptive immunity. Our results also explain key model-specific differences in drug efficacy: when used for drug evaluation, the HDA model begins treatment during the innate immune phase when Mtb are metabolically active, whereas the LDA model initiates therapy after host adaptive immunity has already activated and the bacterial population is immune-constrained. The HDA model is shown to be a mixed model, spanning immune phases with initial treatment acting on replicating bacteria and later treatment acting on immune-constrained populations. Together, the two models offer complementary perspectives on therapeutic activity across the spectrum of bacterial states and provide a framework for designing regimens that are effective against both active and immune-constrained Mtb.

genomics↗

bioRxiv2026

From Sensor Design to Force Maps: A Systematic Evaluation of FRET-based Vinculin Tension Sensors

Mechanical forces transmitted through focal adhesions regulate cell behavior and disease progression, yet remain difficult to quantify at the molecular level. Genetically encoded FRET-based tension probes enable measurements of piconewton-scale forces across specific proteins in living cells, but their quantitative interpretation is highly sensitive to probe design and measurement modality. Here, we systematically compared vinculin tension sensors under identical experimental conditions, evaluating unloaded reference constructs, fluorophore pairs, mechanical sensor modules, and circularly permuted variants. Unloaded controls established a common no-force baseline and validated force-dependent readout. Among the fluorophore pairs tested, the green-red combination Clover-mScarlet-I yielded a higher unloaded FRET efficiency and hence a broader measurable dynamic range. Comparison of six mechanical sensor modules identified the binary-response sensors FL and CC-S2 as the most responsive, showing the largest force-dependent FRET changes and broadest FRET distributions. At the sub-focal adhesion level, CC-S2 reported the steepest proximal-to-distal tension gradient, indicating that vinculin tension increases sharply along peripheral adhesions and exceeds 10 piconewton. Circular permutation experiments revealed that fluorophore orientation has a strong, module-dependent influence on the measured FRET readout. Together, these results establish a comparative framework for interpreting FLIM-based vinculin tension measurements and provide practical design principles for selecting and engineering molecular tension probes.

biophysics↗

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

Antibiotics-induced conformational heterogeneity of a multidrug transporter revealed by single-molecule FRET

Multidrug transporters are membrane proteins that can transport an ensemble of structurally dissimilar compounds and contribute to bacterial multidrug resistance (MDR) by exporting different antibiotics from the cell. However, whether they transport different substrates through a common mechanism or via distinct substrate-dependent mechanisms remains unclear. In this work, we used single-molecule Forster resonance energy transfer (smFRET) to measure time-resolved conformational dynamics of LmrP, a multidrug transporter of the Major Facilitator Superfamily (MFS). We present high-resolution conformational landscapes of LmrP in the presence of different antibiotics. Through multi-parameter Hidden Markov Modeling (mpH2MM), we uncovered transient states and quantified their sub-millisecond interconversion kinetics. We observed antibiotic-dependent heterogeneity in the conformational landscape, both in accessible states and in interconversion rates. Notably, poorly or non-transported antibiotics slow down transition kinetics, pointing to rapid state interconversion as a driver of efficient transport. This suggests that MFS MDR transporters bind and export structurally dissimilar antibiotics by relying on an array of underlying conformational states with ligand-dictated interconversion rates. This work provides novel insights into the mechanism of MDR transporters and advocates for combined structure/dynamics-based drug design when targeting their function.

biophysics↗

bioRxiv2025

Deconvoluting drug interactions based on M. tuberculosis physiologic processes:Transcriptional disaggregation of the BPaL regimen in vivo

Identification of optimal antibiotic combination treatments for tuberculosis (TB) in preclinical studies is impeded by the limited information conventional pharmacodynamic (PD) markers provide about drug interactions. Measurement of individual drug activity based on colony forming units (CFU) does not reliably predict the activity of drug combinations, potentially because one drug may affect the physiology of Mycobacterium tuberculosis (Mtb) in a way that either favors or disfavors the activity of a second drug. SEARCH-TB is a novel candidate PD approach which uses targeted in vivo transcriptional profiling to evaluate the effects of drugs on Mtb physiology. To test the capacity of SEARCH-TB to elucidate drug interactions, we deconstructed the BPaL (bedaquiline, pretomanid, linezolid) regimen in the BALB/c high-dose aerosol mouse infection model, measuring the effect of 2, 7, and 14-day treatment with drugs in monotherapy, pairwise combinations, and as a 3-drug combination. Monotherapy rapidly induced drug-specific Mtb transcriptional responses by day 2 with continued evolution over 14 days. Bedaquiline dominated pairwise combinations with both pretomanid and linezolid. The pretomanid-linezolid combination gave a blended response, inducing transcriptional profiles "intermediate" between either drug. In the 3-drug BPaL regimen, the addition of both pretomanid and linezolid to bedaquiline yielded a greater transcriptional response than expected based on pairwise results. This work demonstrates that physiologic perturbations induced by a single drug may be modified in complex ways when drugs are combined. This establishes proof of concept that SEARCH-TB is a highly granular readout of drug interactions in vivo, providing information distinct from CFU burden and suggesting a future in which regimen selection is informed by in vivo molecular measures of Mtb physiology.

genomics↗

bioRxiv2025

Physiologic recovery of Mycobacterium tuberculosis from drug injury: A molecular study of post antibiotic effect in mice

Post-antibiotic effect (PAE) describes the delayed recovery of bacteria following antibiotic exposure. PAE is thought to underlie tuberculosis (TB) treatment forgiveness, i.e. the capacity of regimens to tolerate non-adherence. The basis of PAE in Mycobacterium tuberculosis (Mtb) remains poorly understood, partly because PAE has conventionally been measured based on change in Mtb burden in vitro rather than change in Mtb physiology in vivo. We investigated Mtb physiologic recovery in the BALB/c mouse model following sub-curative 2- and 4-week durations of the standard isoniazid, rifampin, pyrazinamide, ethambutol (HRZE) treatment. Measurement of rRNA synthesis via the RS ratio(R) and the entire transcriptome via SEARCH-TB elucidated the dynamics of physiologic recovery. Mtb burden did not increase over 28 days of drug-free post-treatment recovery, indicating prolonged PAE in vivo. The RS ratio indicated that Mtb ribosomal RNA synthesis resumed within four days of treatment interruption. However, transcriptional changes indicative of metabolic reactivation were delayed for over two weeks. Processes critical for replication, including expression of genes involved in protein and cell wall synthesis, remained suppressed throughout 28 days post-treatment. Longer treatment induced more extensive physiologic perturbation and was associated with slower and less complete recovery. Expression of processes that are typically induced by environmental stress (e.g., DosR regulon, universal stress proteins, and heat shock proteins) exhibited the reverse, decreasing during drug treatment and rising during recovery. These findings provide a new basis for understanding PAE based on drug-induced injury and physiologic recovery. Following relatively short durations of HRZE, physiologic recovery of Mtb was a slow, sequential and incomplete process in vivo. Our observation that longer treatment resulted in even slower recovery suggests that Mtb may progressively lose capacity to recover. This work establishes a tractable experimental framework for quantifying the forgiveness of new TB treatment regimens in vivo.

microbiology↗

bioRxiv2023

Transcription dosage compensation does not occur in Down syndrome

BackgroundTrisomy 21, also known as Down syndrome, describes the genetic condition of having an extra copy of chromosome 21. The increase in DNA copy number has led to the "DNA dosage hypothesis", which claims that the level of gene transcription is proportional to the genes DNA copy number. Yet many reports have suggested that a proportion of chromosome 21 genes are dosage compensated back towards typical expression levels (1.0x). In contrast, other reports suggest that dosage compensation is not a common mechanism of gene regulation in Trisomy 21, providing support to the DNA dosage hypothesis. ResultsIn our work, we use both simulated and real data to dissect the elements of differential expression analysis that can lead to the appearance of dosage compensation even when compensation is demonstrably absent. Using lymphoblastoid cell lines derived from a family of an individual with Down syndrome, we demonstrate that dosage compensation is nearly absent at both nascent transcription (GRO-seq) and steady-state RNA (RNA-seq) levels. ConclusionsTranscriptional dosage compensation does not occur in Down syndrome. Simulated data containing no dosage compensation can appear to have dosage compensation when analyzed via standard methods. Moreover, some chromosome 21 genes that appear to be dosage compensated are consistent with allele specific expression.

bioinformatics↗

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

Particle-based phasor-FLIM-FRET resolves protein-protein interactions inside single viral particles

Fluorescence lifetime imaging microscopy (FLIM) is a popular modality to create additional contrast in fluorescence images. By carefully analyzing pixel-based nanosecond lifetime patterns, FLIM allows studying complex molecular populations. At the single molecule or single particle level, however, image series often suffer from low signal intensities per pixel, rendering it difficult to quantitatively disentangle different lifetime species, such as during FRET analysis in the presence of a significant donor-only fraction. To address this problem, we combined particle localization with phasor-based FLIM analysis. Using simulations, we first showed that an average of [~]300 photons, spread over the different pixels encompassing single fluorescing particles and without background, is enough to determine a correct phasor signature (standard deviation <5% for a 4 ns lifetime). For immobilized single- or double-labeled dsDNA molecules, we next validated that particle-based phasor-FLIM-FRET readily allows estimating fluorescence lifetimes and FRET from single molecules. Thirdly, we applied particle-based phasor-FLIM-FRET to investigate protein-protein interactions in sub diffraction HIV-1 viral particles. To do this, we first quantitatively compared the fluorescence brightness, lifetime and photostability of different popular fluorescent protein-based FRET probes when genetically fused to the HIV-1 integrase enzyme (IN) in viral particles, and conclude that eGFP, mTurquoise2 and mScarlet perform best. Finally, for viral particles co-expressing FRET-donor/acceptor labeled IN, we determined the absolute FRET efficiency of IN oligomers. Available in a convenient open-source graphical user interface, we believe that particle-based phasor-FLIM-FRET is a promising tool to provide detailed insights in samples suffering from low overall signal intensities. Why it mattersPhasor-FLIM is an extraordinarily popular tool for fluorescence lifetime imaging analysis. However, it remains susceptible for low signal intensities, operational challenges and therefore required informed users and a clear analysis understanding. In this work we developed a convenient all-graphical workflow for quantitative phasor-FLIM in heterogenous and low-signal samples and applied it to quantifying absolute FRET efficiencies from protein-protein interactions inside single viral particles. Moreover, containing a well-illustrated theoretical introduction to time-domain phasor-FLIM, our paper helps novice users to correctly implement phasor-FLIM in standard microscopy practice.

biophysics↗

bioRxiv2023

Application of polystyrene sulfonate (PSS) for inhibiting toxicity of ALS/FTD-linked dipeptide repeats

The GGGGCC (G4C2) expansion in the noncoding region of C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The repeat region is translated into five different dipeptide repeat proteins (DPRs), of which the arginine-rich DPRs (R-DPRs) poly- GR (GRn) and poly-PR (PRn) are highly neurotoxic. In this study, we characterized the protective effect against R- DPR toxicity of polystyrene sulfonate (PSS), an FDA-approved drug applied in hyperkalemia, in biochemical, cellular and iPSC-derived motor neuron (MN) models. We found that PSS, in a length-dependent manner, interacts very tightly with R-DPRs, and releases their bound RNA in R-DPR - RNA mixtures. PSS significantly influences the liquid- liquid phase separation (LLPS) of R-DPRs elicited by RNA and reduces their ensuing cell toxicity in Neuro-2a cells. PSS is cell penetrable, and it can effectively rescue the inhibitory effect R-DPRs on axonal mitochondrial transport in iPSC-derived MNs. Shorter (n < 340) variants of PSS are not toxic either to cells or to mice upon intracerebroventricular injection up to 1 mM concentration. Our results suggest that its polymeric nature endows PSS with an advantageous effect in C9-ALS providing a potential therapeutic tool against this debilitating neurodegenerative disease.

molecular 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

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins

Single-molecule FRET (smFRET) has become an established tool to study biomolecular structure and dynamics in vitro and in live cells. We performed a worldwide blind study involving 19 labs to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems that undergo distinct conformational changes, we obtained an uncertainty of the FRET efficiency of less than {+/-} 0.06, corresponding to an interdye distance precision of [≤] 0.2 nm and accuracy of [≤] 0.5 nm. We further discuss the limits for detecting distance fluctuations with sensitivity down to [lsim] 10% of the Forster distance and provide guidelines on how to detect potential dye perturbations. The ability of smFRET experiments to simultaneously measure distances and avoid averaging of conformational dynamics slower than the fluorescence lifetime is unique for dynamic structural biology.

biophysics↗

bioRxiv2021

Glycine receptor α3K governs mobility and conductance of L/K splice variant heteropentamers

Glycine receptors (GlyRs) are ligand-gated pentameric chloride channels in the central nervous system. GlyR-3 is a possible target for chronic pain treatment and temporal lobe epilepsy. Alternative splicing into K or L variants determines the subcellular fate and function of GlyR-3, yet it remains to be shown whether its different splice variants can functionally co-assemble, and what the properties of such heteropentamers would be. Here, we subjected GlyR-3 to a combined fluorescence microscopy and electrophysiology analysis. We employ masked Pearsons and dual-color spatiotemporal correlation analysis to prove that GlyR-3 splice variants heteropentamerize, adopting the mobility of the K variant. Fluorescence-based single-subunit counting experiments revealed a variable and concentration ratio dependent hetero-stoichiometry. Via single-channel on-cell patch clamp we show heteropentameric conductances resemble those of the 3K splice variant. Our data are compatible with a model where 3 heteropentamerization fine-tunes mobility and activity of GlyR 3 channels, which is important to understand and tackle 3 related diseases. SummaryThe glycine receptor 3 is key to the central nervous systems physiology and involved in chronic pain and epilepsy. In this paper, Lemmens et al. reveal and functionally characterize 3 splice variant heteropentamerization via advanced single-molecule fluorescence image analysis. DeclarationsO_ST_ABSFundingC_ST_ABSWe acknowledge the UHasselt Advanced Optical Microscopy Centre (AOMC). Prof. Em. Marcel Ameloot, the Research Foundation Flanders (FWO, project G0H3716N) and the province of Limburg (Belgium) (tUL Impuls II) are acknowledged for funding the microscopy hardware. V. Lemmens is grateful for a doctoral scholarship from the UHasselt (17DOC11BOF) and KU Leuven (C14/16/053) Special Research Funds (BOF). Conflicts of interest / competing interestsNo conflicts of interest apply. Ethics approvalNot applicable Availability of data and materialAll data and material are available upon request. Code availabilityFluctuation imaging and co-localization analyses were performed in the software package PAM [71]. The software is available as source code, requiring MATLAB to run, or as pre-compiled standalone distributions for Windows or MacOS at http://www.cup.uni-muenchen.de/pc/lamb/software/pam.html or hosted in Git repositories under http://www.gitlab.com/PAM-PIE/PAM and http://www.gitlab.com/PAM-PIE/PAMcompiled. A detailed user manual is available at http://pam.readthedocs.io. Author contributionsConceptualization Meier J.C., Brone B. and Hendrix J.; Investigation and formal analysis Lemmens V. and Thevelein B.; Software development Hendrix J.; Writing the original draft Lemmens V., Thevelein B and Hendrix, J.; Review and editing by all authors.

biochemistry↗

bioRxiv2020

Machine learning-based clustering of nanosized fluorescent extracellular vesicles

Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell communication. The phenotypic profile of EV populations is a promising reporter of disease, with direct clinical diagnostic relevance. Yet, robust methods for quantifying the biomarker content of EV have been critically lacking, and require a single-particle approach due to their inherent heterogeneous nature. Here, we used multicolor single-molecule burst analysis microscopy to detect multiple biomarkers present on single EV. We classified the recorded signals and applied the machine learning-based t-distributed stochastic neighbor embedding algorithm to cluster the resulting multidimensional data. As a proof of principle, we applied the method to assess both the purity and the inflammatory status of EV, and compared cell culture and plasma-derived EV isolated via different purification methods. We then applied this methodology to identify intercellular adhesion molecule-1 (ICAM-1) specific EV subgroups released by inflamed endothelial cells, and to prove that apolipoprotein-a1 is an excellent marker to identify the typical lipoprotein contamination in plasma. Our methodology can be widely applied on standard confocal microscopes, thereby allowing both standardized quality assessment of patient plasma EV preparations, and diagnostic profiling of multiple EV biomarkers in health and disease.

molecular biology↗

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

Substrate binding modulates the conformational kinetics of the secondary multidrug transporter LmrP

The Major Facilitator Superfamily (MFS) is the largest family of secondary active membrane transporters and is found in all domains of Life. MFS proteins are known to adopt different conformational states, yet details on the interconversion rates are crucially needed to understand or target their transport mechanism. Here, we studied the proton/multidrug antiporter LmrP as a model system for antibiotic resistance development in bacteria. The conformational cycle of LmrP is triggered by the protonation of a network of specific amino acids, yet the role of the transported substrate in these transitions has been puzzling. To measure LmrP structure in real-time, we performed solution-based single-molecule Forster resonance energy transfer (smFRET) using a confocal microscope with direct alternating donor/acceptor excitation and multiparameter (intensity, lifetime, anisotropy) detection. Lowering pH from 8 to 5 triggered an overall conformational transition, corroborating that detergent solubilization allows studying the LmrP transport cycle using smFRET. Using a newly developed linear 3-state photon distribution analysis (PDA) model, we show that the apo protein interconverted between two structures at low rate (>>10 ms dwell time) at the cytosolic side while it interconverts dynamically between the 3 states (< 10 ms dwell time) at the extracellular side. When the Hoechst 33342 model substrate is added, inward conformational interconversions are greatly accelerated, coupled to an overall outward conformational halting, consistently with efficient proton exchange with the extracellular environment. Roxithromycin substrate binding did not halt but shift conformational interconversions from one pair of states to another. Substrate dependent structural heterogeneity is indicative of a general mechanism by which MFS transporters can efficiently transport a variety of substrates, and advocates for combined structure/dynamics-based drug design when targeting MDR transporters. BRIEF SUMMARYWe studied the conformational cycle of LmrP, a model for multidrug efflux pumps, using single-molecule Forster resonance energy transfer (smFRET). By following changes in FRET signal between different sets of positions, we specifically investigated how substrate binding modulates structural conversions between inward-open and outward-open states. Using a newly developed probabilistic analysis for describing sequential interconversion kinetics, we show that the apo protein slowly interconverts between defined states at the cytosolic and at the extracellular sides. Binding of the model substrate Hoechst33342 leads to an increase in conformational interconversions at the intracellular side while the extracellular side shows a drastic decrease in conversion, indicating a kinetic uncoupling between both sides. Remarkably, binding of roxithromycin, while also increasing interconversion on the intracellular side, did not slow the extracellular conversions. This indicates that multidrug pumps have evolved substrate-dependent transport mechanisms than enable transport of structurally diverse collection of substrates.

biophysics↗

bioRxiv2020

RNA-dependent structures of the RNA-binding loop in the flavivirus NS3 helicase

The flavivirus NS3 protein is a helicase that has pivotal functions during the viral genome replication process, where it unwinds double-stranded RNA and translocates along the nucleic acid polymer in a nucleoside triphosphate hydrolysis-dependent mechanism. An increased interest in this enzyme as a potential target for development of antiviral therapeutics was sparked by the 2015 Zika virus epidemic in the Americas. Crystallographic and computational studies of the flavivirus NS3 helicase have identified the RNA-binding loop as an interesting structural element, which may function as an origin for the RNA-enhanced NTPase activity observed for this family of helicases. Microsecond-long unbiased molecular dynamics as well as extensive replica exchange umbrella sampling simulations of the Zika NS3 helicase have been performed to investigate the RNA-dependence of this loops structural conformations. Specifically, the effect of the bound single-stranded RNA (ssRNA) oligomer on the putative "open" and "closed" conformations of this loop are studied. In the Apo substrate state, the two structures are nearly isoergonic ({Delta}GO[->]C = -0.22 kcal mol-1), explaining the structural ambiguity observed in Apo NS3h crystal structures. The bound ssRNA is seen to stabilize the "open" conformation ({Delta}GO[->]C = 1.97 kcal mol-1) through direct protein-RNA interactions at the top of the loop. Interestingly, a small ssRNA oligomer bound over 13 [A] away from the loop is seen to affect the free energy surface to favor the "open" structure while minimizing barriers between the two states. The mechanism of the transition between "open" and "closed" states is characterized as are residues of importance for the RNA-binding loop structures. From these results, the loop is hypothesized to be a viable region in the protein for targeted small-molecule inhibition and mutagenesis studies, where stabilization of the "closed" RNA-binding loop will negatively impact RNA-binding and the RNA-enhanced NTPase activity.

biophysics↗