Biology subjects

Reddy, K. D.

Publications and source records attributed to Reddy, K. D..

4 recordsLinked to original sources

bioRxiv2026

Multiscale Free-Energy Methods for Protonation-Coupled Light-Responsive Binding of Ionizable Photoswitchable eDHFR Inhibitors

Photoswitchable ligands enable photocontrol of biomolecular activity by binding to targets in an isomer-dependent, light-responsive manner. Recent developments in ionizable photoswitchable ligands greatly expand their applications but introduce a major design challenge: light-responsive binding can depend on isomeric form, chemical substitution, and binding-induced shifts in protonation equilibria. These effects are tightly coupled, subtle in magnitude, and difficult to predict. Consequently, few computational methods have been developed and systematically benchmarked for quantitatively predicting them. Here, we establish a multiscale free-energy method and benchmark it against experimental data for a series of recently developed photoswitchable inhibitors of Escherichia coli dihydrofolate reductase (eDHFR), a crucial target in photopharmacology. Constant pH replica-exchange molecular dynamics and quantum mechanics/molecular mechanics umbrella sampling quantitatively characterize the ligands protonation-state change upon binding to the eDHFR active site. Thermodynamic integration simulations using alternative alchemical pathways, thermodynamic cycles, and protonation-state assignments were evaluated for predicting light-responsive affinity differentials and substituent effects. Direct cis-to-trans transformations with explicit treatment of environment-dependent protonation states best reproduce experimental trends. Compound-to-compound pathways are less reliable because force-field inaccuracies introduce large pK errors that are difficult to correct when protonation/deprotonation processes implicitly enter the thermodynamic cycle. TI simulations that ignore binding-induced protonation-state changes fail to consistently reproduce experimental trends. Protein-ligand and ligand-water interaction analyses further reveal the energetic and structural origins of isomer-dependent binding. This study establishes a systematic free-energy method for designing ionizable photoswitches in photopharmacology.

pharmacology and toxicology↗

bioRxiv2023

Uncoupled substrate binding underlies the evolutionary switch between Na+ and H+-coupled prokaryotic aspartate transporters

Secondary active membrane transporters harness the energy of ion gradients to concentrate their substrates. Homologous transporters evolved to couple transport to different ions in response to changing environments and needs. The bases of such diversification, and thus principles of ion coupling, are unexplored. Employing phylogenetics and ancestral protein reconstruction, we investigated sodium-coupled transport in prokaryotic glutamate transporters, a mechanism ubiquitous across life domains and critical to neurotransmitter recycling in humans. We found that the evolutionary transition from sodium-dependent to independent substrate binding to the transporter preceded changes in the coupling mechanism. Structural and functional experiments suggest that the transition entailed allosteric mutations, making sodium binding dispensable without affecting ion-binding sites. Allosteric tuning of transporters energy landscapes might be a widespread route of their functional diversification.

biochemistry↗

bioRxiv2022

An environmentally ultrasensitive fluorine-19 NMR probe for monitoring protein conformational equilibria

Limited chemical shift dispersion represents a significant barrier to studying multi-state equilibria of large membrane proteins by 19F NMR. We describe a novel monofluoroethyl 19F probe that dramatically increases the chemical shift dispersion. The improved conformational sensitivity and line shape enable the detection of previously unresolved states in 1D NMR spectra of a 134 kDa membrane transporter. Changes in the populations of these states in response to ligand binding, mutations, and temperature correlate with population changes of distinct conformations in structural ensembles determined by single-particle cryo-electron microscopy. Thus, 19F NMR can guide sample preparation to discover and visualize novel conformational states and facilitate image analysis and 3D classification.

biophysics↗

bioRxiv2021

Heterogeneous substrate binding in a glutamate transporter homologue

Integral membrane glutamate transporters couple the concentrative substrate transport to ion gradients. There is a wealth of structural and mechanistic information about this family. Recent studies revealed transport rate heterogeneity in an archaeal homologue GltPh, inconsistent with simple kinetic models, but its structural and mechanistic determinants remain undefined. In a mutant GltPh, which exclusively populates the outward-facing state, we demonstrate the co-existence of at least two sub-states in slow equilibrium binding the substrate with different apparent affinities. Wild-type GltPh shows similar binding properties, and modulation of the sub-state equilibrium correlates with transport rates. Following binding, the low-affinity sub-state of the mutant is transient. Consistently, cryo-EM on samples frozen within seconds after substrate addition reveals the presence of structural classes with perturbed helical packing of the extracellular half of the transport domain in regions adjacent to the binding site. In contrast, an equilibrated structure does not show such classes. The structure at 2.2 [A] resolution details a pattern of waters in the intracellular half of the domain and resolves classes with subtle differences in the substrate-binding site. We hypothesize that the rigid cytoplasmic half of the domain mediates substrate and ion recognition and coupling, while the extracellular labile half sets the affinity and dynamic properties.

biophysics↗