Biology subjects

Saurabh, S.

Publications and source records attributed to Saurabh, S..

17 recordsLinked to original sources

bioRxiv2026

Multiparametric microenvironment sensing via distinct molecular equilibria in a single cyanine dye

Reading both physical and chemical properties of a microenvironment from a single fluorophore remains a challenge. Here we demonstrate that two coexisting molecular equilibria within one near-infrared cyanine, CyC4, encode two mechanistically distinct ratiometric reporting channels. A meso-amino group and a pendant carboxylate form a tunable intramolecular hydrogen bond that toggles the dye between closed (700 nm) and open (780 nm) emissive conformers. Time-dependent density functional theory (TD-DFT) calculations show that the hydrogen bond raises the LUMO and blue-shifts the emission, establishing the 700/780 emission ratio as a local reporter of hydrogen bonding and polarity. Independently, the chromophore self-associates under crowding- and cosolvent-rich conditions into an aggregate with a blue-shifted, H-type absorption signature near 530-540 nm and a distinct emission near 610 nm upon 540 nm excitation. The intensity of this aggregate band relative to the monomer emission (Ra) serves as a ratiometric reporter of crowding and self-association. Because the two channels arise from distinct molecular equilibria (intramolecular hydrogen bonding vs. intermolecular self-association) they are largely decoupled: a glycerol titration series confirms that the self-association channel (Ra) can be moved while the hydrogen-bonding channel stays essentially fixed. Applied to protein-PEG biomolecular condensates, the two ratios move oppositely with increasing salt, showing that the interior's chemical (polarity, hydrogen bonding) and physical (packing, self-association) environments co-vary across the salt series; a single CyC4 measurement thereby maps this coupled microenvironment, providing a general strategy for multiparametric, ratiometric sensing of crowded microenvironments.

biophysics↗

bioRxiv2026

An aminoglycoside antibiotic drives RNA phase separation

AbstractAminoglycoside antibiotics bind RNA with high affinity through networks of amine and hydroxyl contacts, yet whether this multivalent binding can drive macroscopic RNA phase transitions has never been tested. Here we show that neomycin B (neoB), an FDA-approved aminoglycoside, induces concentration-dependent phase separation of poly(A), poly(U), and total E. coli RNA. Condensate size and density are tunable by pH and ionic strength, which modulate neoB protonation and screening of interdroplet repulsion independently. Comparison with spermine shows that neoB produces more stable condensates despite spermine carrying at least as much effective charge at physiological pH. Fucitol, a hydroxyl-rich polyol lacking amines, fails to condense RNA, showing that cationic amine groups are required and cannot be replaced by hydroxyl groups. Molecular dynamics simulations attribute the greater condensation efficiency of neoB to additional hydrogen bonds donated by its hydroxyl groups. These results reveal that the chemical complexity of aminoglycosides, evolved for tight RNA recognition, also drives a macroscopic RNA phase transition. RNA condensation may contribute to aminoglycoside bactericidal activity and cellular toxicity.

biochemistry↗

bioRxiv2026

A tunable aqueous architecture modulates functionaloutput in biomolecular condensates

Biomolecular condensates organize cellular biochemistry, yet the principles governing their internal solvent architectures remain poorly understood. Most current models focus on macromolecular scaffolds while treating the solvent as a passive, spatially uniform background. Here, we introduce Condensate Spatial Topography via Emission Lifetimes (ConSTEL) to map the continuous solvent polarity landscape inside biomolecular condensates. Using PopZ as a model system, we show that the condensate interior contains a persistent, tunable mosaic of aqueous environments whose apparent polarity, reported by Nile Red fluorescence lifetimes, is organized by thermodynamic state and chemical cues. This microphase-separated solvent architecture defines distinct mesoscale rheological regimes, with intermediate aqueous niches supporting fast, confined tracer motion and highly polar or non-polar extremes forming a slower, viscoelastic mesh. We further demonstrate that drug-like small molecules partition non-uniformly across this landscape according to their physicochemical properties, and that exceeding local solubility limits drives "reciprocal sculpting", in which mismatched guests remodel the host solvent architecture. Together, these results highlight internal solvent organization as an active, tunable determinant of condensate material properties, molecular transport, and partitioning, and suggest that predictive models of condensate function and pharmacology would benefit from incorporating the spatial arrangement of solvent environments alongside bulk composition.

biochemistry↗

bioRxiv2025

Holographic fingerprinting reveals oligomer-driven phase separation in Bovine Serum Albumin

Biomolecular condensates formed through phase separation are fundamental to cellular organization. Although the physical principles underlying intrinsically disordered proteins are well understood, the molecular determinants of condensate formation in globular proteins remain elusive. Here, we employ Holographic Particle Characterization, a label-free, high-throughput imaging technique, to investigate the self-assembly of Bovine Serum Albumin (BSA), a model globular protein. We show that this technique reliably differentiates amorphous aggregates from liquid-like condensates by their distinct refractive indices and morphologies. Coupled with size-exclusion chromatography, our analysis reveals that BSA phase separation strictly depends on higher-order oligomeric assemblies. Monomeric and dimeric fractions fail to form condensates under identical crowding conditions. Furthermore, the internal packing density of these condensates is highly tunable via pH-driven protonation changes but remains insensitive to ionic screening. These findings support a model of "emergent multivalency," where oligomerization creates a structural scaffold enabling hydrophobically stabilized phase separation, thereby defining a molecular threshold for globular proteins condensation.

biochemistry↗

bioRxiv2025

Circadian Rhythms of Whole-Body Metabolite Abundance in Drosophila are Largely Driven by Time of Feeding

Organisms exhibit daily oscillations in metabolite abundance. These oscillations could arise from circadian clock control of metabolic pathways in peripheral tissues, or secondary to rhythmic food intake, which is primarily controlled by central circadian clocks in the brain. To determine the relative contribution of central and peripheral clocks and behavioral cycles to metabolic rhythms in the fruit fly, Drosophila melanogaster, we conducted large-scale metabolite profiling with fine temporal resolution across multiple days in control flies with intact molecular clocks and in flies in which we used the CRISPR/Cas9 gene editing system to specifically eliminate molecular circadian clock function in the fat body, a peripheral metabolic tissue, or the brain. As these latter flies lack feeding rhythms due to central circadian clock dysfunction, we also included an experimental cohort of flies lacking central brain clocks but subjected to time-restricted feeding (TRF) protocols to impose feeding rhythms. Single-nuclei RNA sequencing confirmed selective molecular clock elimination following fat body manipulations, which was associated with predicted alterations in clock gene expression and an attenuation of time-of-day differences in the abundance of fat body transcripts involved in key metabolic pathways. Interestingly, we identified few rhythmically expressed metabolites in flies that were allowed ad libitum food access, and rhythms of metabolite abundance were not drastically altered by tissue-specific molecular clock disruption. In contrast, we found that flies lacking brain clocks but raised on TRF exhibited a profound increase in cyclic metabolites. These findings suggest that whole-body metabolic rhythms in Drosophila are more strongly regulated by feeding cycles than by direct circadian clock control of metabolic pathways despite the presence of metabolic genes that exhibit local-clock dependent modulation of expression across the day.

molecular biology↗

bioRxiv2025

Bacterial Ribonucleoprotein bodies maintain an acidic pH environment as a mechanism of enzyme regulation

Phase separated biomolecular condensates create subcellular niches, yet their role in client regulation remains unclear. Here, we demonstrate that Bacterial Ribonucleoprotein bodies (BR-bodies) are acidic. Using ratiometric fluorescent probes in vivo, we find BR-bodies exhibit a dense-phase pH of [~]5.1, significantly lower than the near-neutral cytoplasm. Single-molecule localization microscopy and fluorescence lifetime imaging reveals that Caulobacter crescentus BR-bodies have spatially variable and acidic nanoscale RNase E clusters. These results question the notion of homogeneous condensates, suggesting that BR-bodies exhibit structural and biochemical diversity, which may facilitate RNA processing under stress. In vitro, pH gradients observed with C-SNARF-4F and RNase E CTD-pHluorin2 deteriorate with increasing buffer concentrations. Notably, the acidic microenvironment within BR-bodies enhances PNPase activity, highlighting the significance of condensate pH regulation. These findings suggest that pH modulation is intrinsic to condensates, directly influencing biochemical reactions and offering a new strategy for designing pH-sensitive drugs to target enzymes within condensates.

microbiology↗

bioRxiv2025

A Mechanical Logic for Bacterial Navigation

Bacterial motility in unconfined liquids is well understood, but many species inhabit crowded, three-dimensional environments where physical constraints shape navigation. Here, we show that the aquatic bacterium Caulobacter crescentus employs backward swimming to overcome confinement, using a force-sensitive mechanism to control directional switching. Using colloidal "{micro}-Traps" to confine single cells within spherical volumes accessible through a narrow aperture, we reveal that cells engage backward motility to enter pores, explore boundaries, and escape confinement. This directional switching is gated by mechanical load: moderate forces on the flagellum promote backward motion, while excessive load suppresses it, preventing futile effort. This tunable, load-sensitive control offers direct evidence for a mechanical logic that governs motility: backward movement is activated when advantageous and disengaged when not. These findings highlight how physical forces influence microbial navigation and adaptation in complex environments.

biophysics↗

bioRxiv2025

Multivalency controls the growth and dynamics of a biomolecular condensate

Biomolecular condensates are essential for cellular organization and function, yet understanding how chemical and physical factors govern their formation and dynamics has been limited by a lack of non-invasive measurement techniques. Conventional microscopy methods often rely on fluorescent labeling and sub-strate immobilization, which can perturb the intrinsic properties of condensates. To overcome these challenges, we apply label-free, contact-free holographic video microscopy to study the behavior of a condensate-forming protein in vitro. This technique enables rapid, high-throughput, and precise measurements of individual condensate diameters and refractive indexes, providing unprecedented insight into size distributions and dense-phase macromolecular concentrations over time. Using this method, we investigate the kinetics of droplet growth, aging, and equilibrium dynamics in the model condensate-forming protein PopZ. By systematically varying the concentration and valence of cations, we uncover how multivalent ions influence condensate organization and dynamics, a hypothesis we further test using super-resolution microscopy. Our findings reveal that PopZ droplet growth deviates from classical models such as Smoluchowski coalescence and Ostwald ripening. Instead, we show that condensate growth is consistent with gelation at the critical overlap concentration. Holographic microscopy offers significant advantages over traditional techniques, such as differential interference contrast (DIC) microscopy, delivering reproducible measurements and capturing condensate dynamics with unparalleled precision. This work highlights the power of holographic microscopy to probe the material properties and mechanistic underpinnings of biomolecular condensates, paving the way for deeper insights into their roles in synthetic systems.

biophysics↗

bioRxiv2024

PEPCy: Photostable fluoromodules for live cell, super-resolution microscopy of surface proteins

We report the evolution and application of two genetically encoded tags that bind the cyanine dyes Cy3 or Cy5 with high specificity and selectivity, in addition to enhancing their photostability. These tags, which we call PEPCy, can be used to target membrane proteins such as G-protein coupled receptors. Due to their orthogonality and high binding-affinity for cognate cyanine dyes, the PEPCy tags can be used for wash-free labeling of cell surface receptors to observe their dynamics at a single molecule level. Together with self-labeling tags, these photostability enhancing proteins against cyanine dyes present a novel, complementary and powerful approach to explore protein dynamics with high spatiotemporal resolution.

biophysics↗

bioRxiv2023

Temperature-driven coordination of circadian transcriptome regulation

The circadian rhythm is an evolutionarily-conserved molecular oscillator that enables species to anticipate rhythmic changes in their environment. At a molecular level, the core clock genes induce a circadian oscillation in thousands of genes in a tissue-specific manner, orchestrating myriad biological processes. While studies have investigated how the core clock circuit responds to environmental perturbations such as temperature, the downstream effects of such perturbations on circadian regulation remain poorly understood. By analyzing bulk-RNA sequencing of Drosophila fat bodies harvested from flies subjected to different environmental conditions, we demonstrate a highly condition-specific circadian transcriptome. Further employing a reference-based gene regulatory network (Reactome), we find evidence of increased gene-gene coordination at low tem-peratures and synchronization of rhythmic genes that are network neighbors. Our results point to the mechanisms by which the circadian clock mediates the flys response to seasonal changes in temperature. Significance StatementThe circadian rhythm enables organisms to anticipate and adapt to changes in their environment. While behavioral changes have been observed in Drosophila melanogaster subjected to low temperatures, little is known regarding how these changes are enacted at a molecular level. By conducting bulk RNA sequencing from fruit flies, we observe that genome-wide circadian oscillation patterns are temperature dependent. Intriguingly, we find that morning and evening peaks of transcriptomic activity shift closer together, consistent with anticipation of a shorter photoperiod in cooler winter weather. We further find that the low-temperature dynamics are highly coordinated with respect to a reference-based gene regulatory network. Our findings provide insights into the mechanisms by which flies adapt to environmental temperature changes.

bioinformatics↗

bioRxiv2023

Intranasal CRMP2-Ubc9 Inhibitor Regulates NaV1.7 to Alleviate Trigeminal Neuropathic Pain

Dysregulation of voltage-gated sodium NaV1.7 channels in sensory neurons contributes to chronic pain conditions, including trigeminal neuropathic pain. We previously reported that chronic pain results in part from increased SUMOylation of collapsin response mediator protein 2 (CRMP2), leading to an increased CRMP2/NaV1.7 interaction and increased functional activity of NaV1.7. Targeting this feed-forward regulation, we developed compound 194, which inhibits CRMP2 SUMOylation mediated by the SUMO-conjugating enzyme Ubc9. We further demonstrated that 194 effectively reduces the functional activity of NaV1.7 channels in dorsal root ganglia neurons and alleviated inflammatory and neuropathic pain. Here, we employed a comprehensive array of investigative approaches, encompassing biochemical, pharmacological, genetic, electrophysiological, and behavioral analyses, to assess the functional implications of NaV1.7 regulation by CRMP2 in trigeminal ganglia (TG) neurons. We confirmed the expression of Scn9a, Dpysl2, and UBE2I within TG neurons. Furthermore, we found an interaction between CRMP2 and NaV1.7, with CRMP2 being SUMOylated in these sensory ganglia. Disrupting CRMP2 SUMOylation with compound 194 uncoupled the CRMP2/NaV1.7 interaction, impeded NaV1.7 diffusion on the plasma membrane, and subsequently diminished NaV1.7 activity. Compound 194 also led to a reduction in TG neuron excitability. Finally, when intranasally administered to rats with chronic constriction injury of the infraorbital nerve (CCI-ION), 194 significantly decreased nociceptive behaviors. Collectively, our findings underscore the critical role of CRMP2 in regulating NaV1.7 within TG neurons, emphasizing the importance of this indirect modulation in trigeminal neuropathic pain.

neuroscience↗

bioRxiv2023

Phosphatase to kinase switch of a critical enzyme contributes to timing of cell differentiation

Cell differentiation is an essential biological process that is often subject to strict temporal regulation. The aquatic bacterium, Caulobacter crescentus, undergoes obligate differentiation from a swarmer cell to a stationary, replication-competent stalked cell, with each cell cycle. Here, we report that the switch from phosphatase to kinase activity of the histidine kinase PleC contributes to timing this differentiation event. We show that PleC PAS domain interaction with the polar scaffold protein PodJ localizes PleC to the cell pole and inhibits in vivo kinase activity. Upon PodJ degradation, released PleC switches to its kinase form and phosphorylates the PleD diguanylate cyclase, initiating the signaling pathway responsible for differentiation. While PodJ inhibits PleC kinase activity, it does not impact PleC phosphatase activity on DivK, which is required for pili biogenesis and flagellar rotation. Thus, PleC PAS domain interaction with PodJ regulates PleC subcellular localization, enzymatic activity, and the timing of cell differentiation, revealing that PAS domains affect enzymatic function on diverse substrates by relying on context dependent binding partners.

developmental biology↗

bioRxiv2020

Modulation of kinase activity within a bacterial membraneless organelle

Biomolecular condensates formed via phase separation enable spatial and temporal organization of enzyme activity. The emergent properties of many condensates have been shown to be responsive to intracellular Adenosine triphosphate (ATP) levels, although the consequences of such mechanisms on enzyme activity are unknown. Here, we show that ATP depletion promotes phase separation in condensates composed of a disordered protein, thereby enhancing the activity of a client kinase enabling robust signaling and maintenance of viability under the stress posed by nutrient scarcity. We propose that a diverse repertoire of condensates can serve as control knobs to tune multivalency and reactivity in response to the metabolic state of bacterial cells. One Sentence SummaryBacterial condensates boost kinase activity under ATP depletion

microbiology↗

bioRxiv2020

A modified ACE2 peptide mimic to block SARS-CoV2 entry

A 23-residue peptide fragment that forms a part of the -1 helix of the ACE2 peptidase domain, the recognition domain for SARS-CoV2 on the ACE2 receptor, holds the potential as a drug to block the viral receptor binding domain (RBD) from forming a complex with ACE2. The peptide has recently been shown to bind the viral RBD with good efficiency. Here, we present a detailed analysis of the energetics of binding of the peptide to the SARS-CoV2 RBD. We use equilibrium molecular dynamics simulation to study the dynamics of the complex. We perform end-state binding energy calculations to gain a residue-level insight into the binding process and use the information to incorporate point mutations into the peptide. We demonstrate using binding energy calculations that the peptide with certain point mutations, especially E17L, shows a stronger binding to the RBD as compared to the wild type peptide. We propose that the modified peptide will thus be more efficient in blocking RBD-ACE2 binding.

biophysics↗

bioRxiv2019

Deconstructing the structural conservation of distantly related bacterial nucleoid-associated proteins using functional chimeras

Nucleoid-associated proteins (NAPs) are DNA-binding proteins critical for the organization and function of the bacterial chromosome. A subclass of NAPs, including Caulobacter crescentus GapR and Escherichia coli H-NS, preferentially bind AT-rich regions of the nucleoid, but phylogenetic groups that encode GapR rarely encode H-NS. Here, utilizing genetic, biochemical, and biophysical studies of GapR in light of a recent DNA-bound crystal structure of GapR (Guo et al, 2018), we show that although evolutionarily distant, GapR and H-NS possess two regions that are structurally and functionally conserved. These regions are involved in self-association and DNA-binding, even though the two proteins oligomerize and regulate transcription differently. Functional analysis of GapR and H-NS protein chimeras identified structural elements present in H-NS but absent in GapR that rationalize differences in transcriptional regulation. In addition, we identified a sequence element unique to GapR that enables assembly into its tetrameric state. Using fluid-atomic force microscopy, we showed that GapR is capable of bridging DNA molecules in vitro. Together, these results demonstrate that two distantly related NAPs utilize evolutionarily conserved structural elements to serve specialized cellular roles via distinct mechanisms.

molecular biology↗

bioRxiv2019

Mesoscale computational protocols for the design of highly cooperative bivalent macromolecules

The last decade has witnessed a swiftly increasing interest in the design and production of novel multivalent molecules as powerful alternatives for conventional antibodies in the fight against cancer and infectious diseases. However, while it is widely accepted that large-scale flexibility (10 - 100 nm) and free/constrained dynamics (100 ns - {micro}s) control the activity of such novel molecules, computational strategies at the mesoscale still lag behind experiments in optimizing the design of crucial features, such as the binding cooperativity (a.k.a. avidity).\n\nIn this study, we introduced different coarse-grained models of a polymer-linked, two-nanobody composite molecule, with the aim of laying down the physical bases of a thorough computational drug design protocol at the mesoscale. We show that the calculation of suitable potentials of mean force allows one to apprehend the nature, range and strength of the thermodynamic forces that govern the motion of free and wall-tethered molecules. Furthermore, we develop a simple computational strategy to quantify the encounter/dissociation dynamics between the free end of a wall-tethered molecule and the surface, at the roots of binding cooperativity. This procedure allows one to pinpoint the role of internal flexibility and weak non-specific interactions on the kinetic constants of the NB-wall encounter and dissociation. Finally, we quantify the role and weight of rare events, which are expected to play a major role in real-life situations, such as in the immune synapse, where the binding kinetics is likely dominated by fluctuations.\n\nSIGNIFICANCEMultivalent and multispecific molecules composed of polymer-linked nanobodies have gained interest as engineered alternatives to conventional antibodies. These therapeutic molecules have a larger reach due to their smaller size and promise substantial and tunable gains in avidity. This paper studies a model diabody to lay the bases of a multi-scale computational design of the structural and dynamical determinants of binding cooperativity, rooted in a blend of atomistic and coarse-grained MD simulations and concepts from statistical mechanics.

biophysics↗

bioRxiv2017

Genetically Targeted Ratiometric and Activated pH Indicator Complexes (TRApHIC) for Receptor Trafficking

Fluorescent protein based pH sensors are a useful tool for measuring protein trafficking through pH changes associated with endo-and exocytosis. However, commonly used pH sensing probes are ubiquitously expressed with their protein of interest throughout the cell, hindering the ability to focus on specific trafficking pools of proteins. We developed a family of excitation-ratiometric, activatable pH responsive tandem dyes, consisting of a pH sensitive Cy3 donor linked to a fluorogenic malachite green acceptor. These cell-excluded dyes are targeted and activated upon binding to a genetically expressed fluorogen activating protein, and are suitable for selective labeling of surface proteins for analysis of endocytosis and recycling in live cells using both confocal and superresolution microscopy. Quantitative profiling of the endocytosis and recycling of tagged {beta}2-adrenergic receptor (B2AR) at a single vesicle level revealed differences among B2AR agonists, consistent with more detailed pharmacological profiling.

cell biology↗