Biology subjects

Pingali, S. V.

Publications and source records attributed to Pingali, S. V..

4 recordsLinked to original sources

bioRxiv2026

Conformational Reorganization of a Processive Cellulase on Crystalline Cellulose

Enzymes that degrade insoluble polymers must couple catalytic chemistry to productive engagement with heterogeneous substrate interfaces, yet direct structural information on these interfacial states remains limited. We investigated the conformational organization of the processive cellulase Trichoderma reesei Cel7A both free in solution and bound to an insoluble cellulose substrate using small-angle neutron scattering (SANS). Use of biosynthetically deuterated bacterial cellulose allowed the scattering contribution of the cellulose-associated Cel7A holoenzyme to be selectively highlighted using SANS with contrast matching. SANS measurements performed under conditions supporting high or low catalytic activity revealed distinct differences between the free and cellulose-bound enzyme. Under poorly active conditions, the conformational distributions of free and bound Cel7A were similar. In contrast, catalytically favorable conditions produced an additional interdomain correlation in the bound enzyme, consistent with redistribution toward a substrate-selected conformational ensemble. Molecular dynamics simulations showed that free Cel7A samples can assume compact, intermediate, and extended conformations, but these solution-state ensembles did not reproduce the structural features of cellulose-bound Cel7A. These results identify productive cellulose engagement as a structurally distinct state of the Cel7A holoenzyme and support a dynamic model in which the linker and carbohydrate-binding module modulate interfacial positioning of the catalytic domain rather than functioning as a rigid mechanical unit during processive hydrolysis. More broadly, the findings suggest that heterogeneous biocatalysis depends not only on substrate binding and active-site chemistry, but also on interface-selected conformational states that determine whether binding becomes productive. The study further demonstrates how deuteration and neutron contrast variation can reveal functional macromolecular conformations directly within complex interfacial environments.

biochemistry↗

bioRxiv2026

Integrating Segmental Deuteration iCM-SANS with SAXS and MD for Dynamical Analysis of Multi-domain Proteins

Multi-domain proteins (MDPs) adopt diverse conformations arising from cooperative inter-domain motions, and such dynamics are intimately coupled to their biological functions. Quantitative characterization of these motions is crucial for elucidating their functional mechanisms. Although small-angle X-ray scattering (SAXS) provides information on overall domain arrangement, the limited experimental constraints hinder reliable discrimination of conformational ensembles derived from molecular dynamics (MD) simulations. To address this limitation, complementary experimental constraints that enable to observe domain-selective structural information are required. Inverse contrast-matching small-angle neutron scattering (iCM-SANS), combined with segmental deuteration, enables selective visualization of individual domains and thus provides such complementary information. However, practical strategies for preparing segmentally deuterated MDPs with arbitrary domain labelling have yet to be established. Here, we develop an experimental protocol that integrates controlled protein deuteration with high-efficiency multi-step protein ligation to generate a segmentally deuterated MDP in high yield. The combined use of SAXS and iCM-SANS yields complementary structural constraints that enhance discrimination of MD-derived conformational ensembles. This protocol expands the applicability of segment-selective visualization and also provides an opportunity for high-precision analysis of dynamics in complex MDPs. SynopsisSegmental deuteration enabled by high-efficiency multi-step protein ligation, combined with inverse contrast-matching SANS and SAXS, provides structural constraints that improve discrimination of molecular dynamics ensembles of multi-domain proteins. IMPORTANTthis document contains embedded data - to preserve data integrity, please ensure where possible that the IUCr Word tools (available from http://journals.iucr.org/services/docxtemplate/) are installed when editing this document.

biophysics↗

bioRxiv2020

Carbohydrate binding domains facilitate efficient oligosaccharides synthesis by enhancing mutant catalytic domain transglycosylation activity

Chemoenzymatic approaches using carbohydrate-active enzymes (CAZymes) offer a promising avenue for synthesis of glycans like oligosaccharides. Here, we report a novel chemoenzymatic route for cellodextrins synthesis employed by chimeric CAZymes, akin to native glycosyltransferases, involving the unprecedented participation of a non-catalytic lectin-like or carbohydrate-binding domains (CBMs) in the catalytic step for glycosidic bond synthesis using {beta}-cellobiosyl donor sugars as activated substrates. CBMs are often thought to play a passive substrate targeting role in enzymatic glycosylation reactions mostly via overcoming substrate diffusion limitations for tethered catalytic domains (CDs) but are not known to participate directly in any nucleophilic substitution mechanisms that impact the actual glycosyl transfer step. Our study provides evidence for the direct participation of CBMs in the catalytic reaction step for {beta}-glucan glycosidic bonds synthesis enhancing activity for CBM-based CAZyme chimeras by >140-fold over CDs alone. Dynamic intra-domain interactions that facilitate this poorly understood reaction mechanism were further revealed by small-angle X-ray scattering structural analysis along with detailed mutagenesis studies to shed light on our current limited understanding of similar transglycosylation-type reaction mechanisms. In summary, our study provides a novel strategy for engineering similar CBM-based CAZyme chimeras for synthesis of bespoke oligosaccharides using simple activated sugar monomers.

biochemistry↗

bioRxiv2019

CELLULOSE SYNTHASE INTERACTIVE1- and Microtubule-Dependent Cell Wall Architecture Is Required for Acid Growth in Arabidopsis Hypocotyls

Auxin-induced cell elongation relies in part on the acidification of the cell wall, a process known as acid growth that presumably triggers expansin-mediated wall loosening via altered interactions between cellulose microfibrils. Cellulose microfibrils are a major determinant for anisotropic growth and they provide the scaffold for cell wall assembly. Little is known about how acid growth depends on cell wall architecture. To explore the relationship between acid growth-mediated cell elongation and plant cell wall architecture, two mutants (jia1-1 and csi1-3) that are defective in cellulose biosynthesis and cellulose microfibril organization were analyzed. The study revealed that cell elongation is dependent on CSI1-mediated cell wall architecture but not on the overall crystalline cellulose content. We observed a correlation between loss of crossed-polylamellate walls and loss of auxin- and fusicoccin-induced cell growth in csi1-3. Furthermore, induced loss of crossed-polylamellate walls via disruption of cortical microtubule mimics the effect of csi1 in acid growth. We hypothesize that CSI1- and microtubule-dependent crossed-polylamellate walls are required for acid growth in Arabidopsis hypocotyls.

plant biology↗