Biology subjects

Prodhan, S.

Publications and source records attributed to Prodhan, S..

3 recordsLinked to original sources

bioRxiv2026

Secondary nucleation drives polymorph diversity in hIAPP amyloids

Amyloid fibrils are implicated in a myriad of human diseases. A striking observation is that fibrils extracted from diseased tissues are characterized by a restricted set of folds unique to the specific pathology. In contrast, fibrils grown \textit{in vitro} exhibit extensive structural diversity, suggesting that specific environmental and biochemical mechanisms \textit{in vivo} enforce structural selectivity. Here, we combine two-dimensional infrared (2D IR) spectroscopy and cryo-electron microscopy (cryo-EM) to investigate the mechanisms governing polymorph formation in the human Islet Amyloid Polypeptide (hIAPP). We demonstrate that 2D IR can resolve populations of distinct polymorphs identified by cryo-EM, enabling rapid label-free screening of conditions prior to labor-intensive microscopy screening. We find that conditions favoring secondary nucleation, such as high protein concentration, increase polymorphic diversity. Crucially, cryo-EM reveals that formed by secondary nucleation do not structurally replicate the parent template. Finally, by selectively inhibiting secondary nucleation using the C-terminal domain of the DNAJB6 chaperone, we steer aggregation toward a monomorphic state. These findings highlight the critical role of molecular chaperones in fibril polymorph selection.

biophysics↗

bioRxiv2025

Chemical mechanism of allosteric and asymmetric dark reversion in a bacterial phytochrome uncovered by cryo-EM.

Phytochromes are light-sensitive proteins found in plants, fungi, and bacteria. They exist in two functional states, Pr and Pfr, distinguished by Z/E isomers of their bilin chromophore. The chromophore can photoswitch between these states, but also thermally converts in darkness. Despite the importance of the latter reaction, it remains unclear how it is controlled by the phytochrome. Here, we present single-particle cryo-EM measurements on the Pseudomonas aeruginosa bacteriophytochrome (PaBphP) carried out at multiple time points during dark reversion from Pr to Pfr. These experiments resolve the structure of a PrPfr hybrid state. Surprisingly, we find that only protomer B converts back to Pfr in the hybrid, while protomer A remains in Pr. We identify structural asymmetries in the precursor Pr state, which extend from the homodimer interface to a conserved histidine (H277). The hydrogen-bonding network around the chromophore is modulated, explaining how the phytochrome gains control over the activation energy of the isomerization reaction. These findings establish that dark reversion is governed by conformational selection between two substates, whereby one is "dark-reversion ready" and the other one blocks the reaction. Moreover, we explain how the equilibrium of the states is allosterically controlled across the dimer. Together, these findings provide a structural framework for tuning phytochrome signaling lifetimes in optogenetic applications. Significance statementThe dark reversion reaction of phytochromes is crucial to their signalling role in plants, bacteria, and fungi, but it is vastly understudied in terms of its chemical mechanism. It remains unsolved how the reaction can proceed at all, given that the activation energy is prohibitively high for the isomerization to occur in solution. Using time-resolved cryo-EM, we present a chemical and structural framework for understanding how the protein binding pocket regulates the reaction. Our results establish that conformational selection between two substates controls dark reversion, providing a rare example of strongly asymmetric reactivity across a dimeric protein. This opens the way for rational engineering of the lifetimes of the signaling states in phytochromes.

biochemistry↗

bioRxiv2025

Detection of a hybrid PrPfr state in the dark reversion of abathy phytochrome indicates inter-dimer allostery.

Phytochromes are photosensor proteins which detect light in plants, fungi, and bacteria. They photoswitch between red light absorbing (Pr) and far-red light absorbing (Pfr) states, however, thermal reversion in the dark is an equally important factor in controlling their signaling levels. Phytochromes are generally dimeric proteins, and mixed PrPfr states are therefore possible. These states have been implied in the dark reversion studies of plant phytochromes, but not in bacterial phytochromes. Here, we investigate the dark reversion kinetics of the bathy phytochrome from P. aeruginosa (PaBphP) using UV-Vis absorption spectroscopy. A single set of time-resolved spectra does not conclusively reveal the presence of a mixed PrPfr state, as both a direct Pr[->] Pfr model or a sequential Pr[->] PrPfr[->] Pfr model fit the spectral kinetics. However, a systematic analysis of dark reversion kinetics with varying Pr/Pfr ratios can only be satisfactorily fit by the sequential model, which indicates the presence of an intermediate PrPfr state. A newly designed monomeric variant of PaBphP provides strong support for this interpretation. Temperature-dependent kinetics revealed similarly low activation energies for the dark reversion processes of both proteins, consistent with a previously proposed keto-enol tautomerization preceding dark reversion. Interestingly, our results suggest allosteric regulation of dark reversion across the dimer, which we propose to be a contributing factor in phytochrome signaling.

biochemistry↗