Biology subjects

Bhunia, A.

Publications and source records attributed to Bhunia, A..

4 recordsLinked to original sources

bioRxiv2026

Mechanistic insights into proteolytic activation ofmembrane-bound NAC transcription factor SlNACMTF3 intomato plants under heat stress

Stress is perceived at the cellular membrane, where membrane-bound transcription factors (MTFs) are activated, initiating a rapid gene-regulation cascade. We previously demonstrated the multifaceted role of tomato MTF, SlNACMTF3, in regulating expression of biotic and abiotic stress responsive genes. However, the molecular mechanism underlying its activation following stress perception remained unknown. Here, we show that the membrane rhomboid protease SlRP3 recognizes an ISK motif near the transmembrane domain of SlNACMTF3 for cleavage and releasing the transcription factor from the membrane. SlRP3 and SlNACMTF3 co-localize within the same plasma membrane microdomain and interact directly. Molecular dynamics simulations supported the proposed mechanism, in which heat-induced conformational switching of SlRP3, driven by reorientation of the L5 loop, enhances substrate accessibility and proteolytic activity. Furthermore, mutation of key SlRP3 residues involved in substrate interaction impaired SlNACMTF3 activation and reduced the expression of stress33 responsive genes. Analysis with SlNACMTF3-overexpressing tomato lines and SlRP3 transient knockdown leaves further revealed that transcription factor activation, stress35 responsive gene regulation, and cellular ROS detoxification occur coordinately. Collectively, these findings provide mechanistic insight into stress-triggered SlNACMTF3 activation and identify SlRP3 as a novel membrane-associated stress sensor that activates a major transcriptional regulator to enhance stress preparedness in economically important tomato plants.

plant biology↗

bioRxiv2020

Mechanistic Studies of the Stabilization of Insulin Helical Structure by Coomassie Brilliant Blue

Human insulin (HI) is an essential protein hormone and its biological activity mostly depends on folded and active conformation in the monomeric state. The present investigation established that Coomassie Brilliant Blue G-250 (CBBG), a small multicyclic hydroxyl compound can reversibly bind to the hormonal protein dimer and maintained most of -helical folds crucial for biological function of the enzyme. The solution-state 1D NMR and isothermal calorimetric analysis showed a sub-micromolar binding affinity of the molecule to HI. 2D NOESY NMR established that the HI dimer undergoes residue level local conformational change upon binding to CBBG. The chemical shift perturbation and the NOE parameters of active protons of amino acid residues throughout the polypeptides further suggested that CBBG upon binding the protein stabilize -helixes of both the A and B subunits of the hormonal protein. The changes in Gibbs free energy ({triangleup}G) of the binding was of ~-11.1 kcal/mol and suggested a thermodynamically favourable process. The changes in enthalpy ({triangleup}H) and entropy term (T{triangleup}S) were -57.2 kcal/mol and -46.1 kcal/mol, respectively. The negative changes in entropy and the NOE transfer effectiveness of several residues in the presence of CBBG molecules indicated that the binding was an enthalpy driven favourable equilibrium process. The NMR-based atomic resolution data and molecular docking studies confirmed that the CBBG binds to HI at the dimeric stage and prevents the availability of the crucial residue segments that partake directly in further oligomerization and subsequent fibrillation. Extended computational analysis based on chemical shift perturbation of protons of active residues further established receptor-ligand based pharmacophore model comprised of 5 hydrophobic and a hydrogen bond acceptor features that can anchor the residues at the A and B chains of HI and inhibit the partial unfolding and hydrophobic collapse to nucleate the fibrillation. Taken together, the results demonstrated that CBBG and their close analogues might be useful to develop a formulation that will maintain the active and functional form of the hormonal protein for a significantly longer time. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/267799v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@b2c363org.highwire.dtl.DTLVardef@39b7b6org.highwire.dtl.DTLVardef@14c4d71org.highwire.dtl.DTLVardef@704f00_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2019

High resolution structure of a partially folded insulin aggregation intermediate

Insulin has long served as a model for protein aggregation, both because of the importance of aggregation in insulin manufacture and because the structural biology of insulin has been extensively characterized. Despite intensive study, details about the initial triggers for aggregation have remained elusive at the molecular level. We show here that at acidic pH, the aggregation of insulin is likely initiated by a partially folded monomeric intermediate whose concentration is controlled by an off-pathway micellar species. High resolution structures of the partially folded intermediate show that it is coarsely similar to the initial monomeric structure but differs in subtle details - the A chain helices on the receptor interface are more disordered and the B chain helix moves away from C-terminal A chain helix. The result of these movements is the creation of a hydrophobic cavity in the center of the protein that may serve as nucleation site for oligomer formation. Knowledge of this transition may aid in the engineering of insulin variants that retain the favorable pharamacokinetic properties of monomeric insulin but are more resistant to aggregation.

biophysics↗

bioRxiv2018

Non-productive Binding Modes as a Prominent Feature of Aβ1-40 Fiber Elongation: Insights from Molecular Dynamics Simulation

AbstractAmyloid formation has been implicated in a number of neurodegenerative diseases. The elongation of amyloid fibers is thermodynamically strongly favorable but kinetic traps exist where the incoming monomer binds in an incompatible conformation that blocks further elongation. Unfortunately, this process is difficult to follow experimentally at the atomic level. It is also too complex to simulate in full detail and thus so far has been explored either through coarse-grained simulations, which may miss many important interactions, or full atomic simulations in which the incoming peptide is constrained to be near the ideal fiber geometry. Here we use an alternate approach starting from a docked complex in which the monomer is from an experimental NMR structure of one of the major conformations in the unbound ensemble, a largely unstructured peptide with the central hydrophobic region in a 310 helix. A 1000 ns full atomic simulation in explicit solvent shows the formation of a metastable intermediate by sequential, concerted movements of both the fiber and monomer. A Markov state model shows the unfolded monomer is trapped at the end of the fiber in a set of interconverting anti-parallel {beta}-hairpin conformations. The simulation here may serve as a model for the binding of other non-{beta}-sheet conformations to amyloid fibers.

biophysics↗