Biology subjects

Purohit, A.

Publications and source records attributed to Purohit, A..

3 recordsLinked to original sources

bioRxiv2025

Structural and Functional Mechanisms Underlying Activation Gate Dynamics and IFM Motif Accessibility in Human Nav1.5

Voltage-gated sodium channels are vital for regulating excitability in muscle and nerve cells, and their dysregulation is linked to a range of diseases. However, therapeutic targeting of Nav channels remains challenging due to a limited understanding of their gating mechanisms. Here, we present a cryo-EM structure of human Nav1.5 in an intermediate open state, stabilized by interactions between the N-terminal domain and the S6I segment. This structure reveals a previously uncharacterized Na+ binding site adjacent to the conserved inactivation (IFM) motif. Molecular dynamics simulations demonstrate that monovalent cations stably occupy this site, while electrophysiological recordings demonstrate that ion binding modulates IFM motif docking and fast inactivation kinetics. Our findings reveal that IFM accessibility is dynamically regulated in this intermediate state, challenging the canonical hinged-lid model of fast inactivation. Collectively, our study provides a revised structural framework for Nav1.5 gating mechanisms, suggesting an alternative pathway for ion accessibility that may inform better mechanistic and therapeutic strategies for treating Nav1.5-related cardiac arrhythmias.

biophysics↗

bioRxiv2025

PYEAST - Python Enabled Automated Strain Transformaiton

Saccharomyces cerevisiae is a widely used biotechnological workhorse in both academic and industrial settings. One reason for its continued popularity is the extensive legacy of genetic tools, developed over its long history of use, that enable precise manipulation of the S. cerevisiae genome. These tools have enabled extensive genetic characterisation and dramatic re-programming efforts for applications ranging from fundamental research to industrial chemical production. Here we present a digital toolkit called PYEAST (Python Enabled Automated Strain Transformation) that encodes some of the most widely used methods for working with S. cerevisiae and modernizes them to leverage advances in DNA synthesis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/655004v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@4584feorg.highwire.dtl.DTLVardef@1e65caorg.highwire.dtl.DTLVardef@1acbe2borg.highwire.dtl.DTLVardef@1f91abc_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2020

Potassium Glutamate and Glycine Betaine Induce Self-Assembly of Sliding Clamps into Higher Order Oligomers

Sliding clamps are oligomeric ring-shaped proteins that increase the efficiency of DNA replication. The stability of the Escherichia coli {beta}-clamp, a homodimer, is particularly remarkable. The dissociation equilibrium constant of {beta} is of the order of 10 pM in buffers of moderate ionic strength. Coulombic electrostatic interactions have been shown to contribute to this remarkable stability. Increasing NaCl concentration in the assay buffer results in decreased dimer stability and faster subunit dissociation kinetics in a way consistent with simple charge-screening models. Here, we examine non-Coulombic ionic effects on the oligomerization properties of sliding clamps. Replacing NaCl by KGlu, the primary cytoplasmic salt in E. coli, results in the formation of assemblies that involve two or more rings stacked face-to-face. Results can be quantitatively explained on the basis of unfavorable interactions between KGlu and the functional groups on the protein surface, which drive biomolecular processes that bury exposed surface. Similar results were obtained with the S. cerevisiae PCNA sliding clamp, suggesting that KGlu effects are not specific to {beta}. Clamp association is also promoted by glycine betaine, a zwitterionic compound that accumulates intracellularly when E. coli is exposed to high concentrations of extracellular solute. Possible biological implications are discussed.

biophysics↗