Biology subjects

Shankar, M. K.

Publications and source records attributed to Shankar, M. K..

4 recordsLinked to original sources

bioRxiv2026

Covalent bond formation caught in a LOV photoreceptor

Light-oxygen-voltage (LOV) domains are blue-light photoreceptors of plants, algae and fungi, and among the most widely used tools in optogenetics. They switch on by forming a covalent thioether bond between a conserved cysteine and their flavin chromophore, in a reaction that needs a proton to cross from the cysteine to the flavin through a pocket containing essentially no water. Its mechanism has been debated for two decades1, and because the chemistry is over within a microsecond its elementary steps have stayed hidden. Here we combine 10 time-resolved serial femtosecond crystallography snapshots and infrared spectroscopy with QM/MM calculations to resolve the entire sequence of events at 1.4 [A] resolution: from excited-state distortion of the flavin ring (10-100 ps), through hydration of a surface channel (10 ns) and a single ordered water reaching the active site as the reactive cysteine shifts between its conformations (100-500 ns), to the thioether bond itself, caught half-formed at 1 {micro}s (half the molecules reacted, half still poised) and complete at 10-100 {micro}s. That water bridges the cysteine and the flavin and shuttles the proton, lowering the barrier from [~]35 to [~]15 kcal/mol and accelerating the reaction by roughly fourteen orders of magnitude (without it, the half-life would be [~]237,000 years), then departs before the bond forms. Proteins can therefore hydrate a dehydrated active site transiently and on demand to overcome otherwise prohibitive reaction barriers, a catalytic strategy that reaches well beyond photoreceptors.

biophysics↗

bioRxiv2026

Serial femtosecond crystallography reveals the pH-driven allosteric mechanism of hexamer glargine

Insulin glargine is formulated at acidic pH but acts after transferring to near-neutral tissue, where its prolonged effect is commonly attributed to isoelectric depot formation. However, the structural pathway linking precipitation to delayed release has remained unresolved. Here we combine ambient-temperature serial femtosecond crystallography, solution biophysics, and multiscale network analyses to define the pH-dependent conformational landscape of hexameric glargine across pH 8.4, 7.3, 6.4, and 5.1. We resolve full hexameric glargine structures and identify a previously unreported, pH-coupled lattice transition from P1211 (near-neutral) to R3:H (acidic), accompanied by redistribution from compact phenolic Rf6-state assemblies to more plastic yet structurally coherent TRf/T3Rf3 states. This transition is accompanied by B-chain N-terminal unpeeling, phenol-pocket collapse, hydration loss, and electrostatic rewiring, and is mirrored in solution by oligomeric heterogeneity, Raman amide-I broadening, reduced thermal stability, and a blue-shifted intrinsic fluorescence maximum. Multiscale analyses further indicate that acidification does not create a new dynamical regime but reweighs pre-existing collective modes along a continuous free-energy landscape. These results support a revised mechanism in which isoelectric precipitation and delayed dissociation are mechanistically coupled through structurally organized molten-like intermediate states, linking glargine pharmacology to intrinsic allosteric redistribution within the hexamer. These findings establish a structural blueprint for benchmarking biosimilar glargine and for engineering next-generation basal insulins by tuning allosteric plasticity and intermediate-state stability.

biophysics↗

bioRxiv2026

Reciprocal-space mapping of diffuse scattering by serial femtosecond crystallography reveals analog-specific disorder in insulin analogs

Insulin detemir and insulin aspart are clinically complementary analogs engineered for distinct pharmacokinetic behavior, yet their comparative structural heterogeneity across temperature regimes remains insufficiently resolved. Here, we present a multi-scale crystallographic analysis integrating near-physiological serial femtosecond crystallography (SFX) with previously reported cryogenic and ambient multicrystal datasets for both analogs. Across conventional quality metrics, reciprocal-space intensity-field reconstructions, model-derived diffuse-scattering representations, Ramachandran stereochemical validation, solvent-accessibility coupling (SAArea-MSArea), and residue-level BDamage (a packing-normalized B-factor metric highlighting local mobility outliers) profiling, we identify a coherent ambient-versus-cryogenic contrast. Ambient datasets show broader reciprocal-space heterogeneity and more diffuse model-space distributions, consistent with increased conformational sampling outside cryogenic trapping. Despite this shared trend, disorder partitioning is analog-specific: detemir exhibits strong pseudo-translational signatures with moderate twinning, whereas aspart shows weak pseudo-translation but pronounced merohedral twinning approaching the theoretical twinned limit in ambient conditions. Importantly, backbone stereochemistry remains globally stable across all datasets, indicating that the observed differences reflect structured heterogeneity rather than model deterioration. Collectively, these findings support an ensemble-aware interpretation of insulin crystallography and provide transferable structural descriptors for analog comparison, stability assessment, and formulation-oriented design.

biophysics↗

bioRxiv2024

Crystal structure of a MarR family transcriptional regulator protein

The multiple antibiotic resistance regulator (MarR) family of transcription factor proteins form a large group of multitasking bio-molecules in pathogenic Escherichia coli (E. coli). HosA is one of these MarR transcription factors reported in dozens of pathogenic E. coli with highly conserved sequence profiles. The HosA from the enteropathogenic E. coli O127:H6 (strain E2348/69), a predominantly monomeric protein, was overexpressed in E. coli and purified. The HosA protein crystals were obtained in microbatch under oil method at 4{degrees} C. The X-rays of the diffracted spots were extended to 2.21 [A] resolution. The crystal belongs to the space group P4322, with unit-cell parameters a = 67.16 [A], b = 67.16 [A], c = 95.66 [A] and = {beta} = {gamma} = 90{degrees}. In the asymmetric unit, monomeric HosA protein was crystallized and confirmed with the Matthew coefficient analysis (3.48 [A]3 Da-1). The monomeric structure is compared with previously solved structures of other homologous transcription factors. This confirmed the winged loop at the DNA binding region of the HosA protein.

biophysics↗