Biology subjects

Chundawat, S. P. S.

Publications and source records attributed to Chundawat, S. P. S..

At least 19 recordsLinked to original sources

bioRxiv2026

N-GLYcanyzer monitoring of a microperfusion process reveals critical parameters' impact on Trastuzumab biosimilar N-glycoform time evolution

Controlling monoclonal antibody (mAb) N-glycosylation as a critical quality attribute (CQA) remains challenging in continuous and intensified biomanufacturing, where conventional end-point analyses obscure dynamic pathway responses. Here, we deployed the N-GLYcanyzer process analytical technology (PAT) for automated monitoring of a microperfusion process that reveals critical parameters' impact on Trastuzumab biosimilar N-glycoform time evolution across a 20-day culture duration of Chinese Hamster Ovary (CHO) cells in a ~2 mL scale-down microbioreactor perfusion platform. Using offline PAT assays combined with our integrated at-line PAT workflow, we systematically screened six perfusion process parameters: carbon source, viable cell density (VCD), perfusion rate, temperature, and pH. While nutrient limitation and thermal/acidic stress significantly disrupted N-glycans maturation, notably triggering high-mannose (Man5) accumulation and severe productivity penalties under pH 6.7 and 39 {degrees}C operation, targeted feed supplementation with galactose and MnCl2 consistently shifted glycoform profiles away from G0F toward galactosylated species (G1F, G1F', G2F). Crucially, coupling this combined feed strategy with an intensified perfusion rate (1.2 VVD) achieved a sustained, desired galactosylation shift without a productivity cost. These findings demonstrate that nutrient feed composition and perfusion rate are primary levers for steering continuous intensified microperfusion processes for biologics production with desired N-glycoform states and highlight the utility of automated at-line PAT like N-GLYcanyzer for enabling real-time attribute-based control in continuous biomanufacturing.

bioengineering↗

bioRxiv2026

Force-induced unfolding versus unbinding of a cellulosomal carbohydrate-binding module from cellulose interface

Carbohydrate-binding modules (CBMs) anchor microbial cellulosomes to cellulose surfaces, aiding biomass degradation under mechanical stress. To understand how mechanical pulling affects this process, we combine steered molecular dynamics (SMD) simulations with acoustic force spectroscopy (AFS) to investigate Clostridium thermocellum CBM3a unbinding from cellulose under different pulling configurations (C- versus N-terminus). Our SMD simulations reveal that C-terminal pulling induces substantial CBM3a unfolding prior to unbinding, whereas N-terminal pulling results in detachment with the protein largely intact. Unbinding proceeds via two distinct mechanisms across the planar binding motif: a TRP mode (initiating via TRP135) and a TYR mode (initiating via TYR84). AFS experiments corroborate the SMD findings, showing that C-terminal pulling yields lower rupture forces and a 7.5-fold higher unbinding off-rate compared to N-terminal pulling of CBM3a from cellulose. These results demonstrate that structural asymmetry and pulling orientation critically govern the mechanical stability, unfolding pathways, and cellulose-binding dynamics of cellulosomal proteins.

biophysics↗

bioRxiv2026

Chemical Rescue Serves as a Predictive Proxy for Glycosynthase Activity on Glycosidic Bonds via a Shared Glycosyl Oxocarbenium Transition State

Engineered glycosynthases (GSs) are powerful biocatalysts for custom glycan synthesis, yet their optimization via directed evolution is severely constrained by bottlenecks in high-throughput screening for activated azido-sugar donors. Here, we demonstrate that chemical rescue (CR)--the azide-mediated restoration of hydrolytic activity in nucleophile-deficient mutants--serves as a predictive, high-throughput proxy for glycosynthase activity. Applying an azide-responsive Escherichia coli biosensor screen to a site-saturation mutagenesis library of Thermotoga maritima -L-fucosidase (TmAfc), we established a strong rank-order correlation between CR and GS activities in both crude lysates ({rho} = 0.73) and purified enzymes ({rho} = 0.95). Transition path sampling and QM/MM umbrella sampling revealed that both pathways proceed through a shared oxocarbenium-ion-like transition state ({Delta}G{ddagger} {approx} 8.7 kcal/mol), providing a structural and thermodynamic rationale for using CR to select for transition-state-stabilizing mutations. Biochemical characterization of top-performing variants yielded an engineered fucosynthase (TmAfc_D224G_N70D_T392S) exhibiting a nearly 100-fold enhancement in Vmax alongside altered regioselectivity. This two-tiered screening framework leverages cost-effective chemical rescue assays to streamline glycosynthase engineering for tailored glycans synthesis.

biochemistry↗

bioRxiv2026

Supercharged binding modules can modulate engineered poly(ethylene terephthalate) hydrolase thermostability and functional persistence

Poly(ethylene terephthalate) (PET) is a highly recalcitrant polyester plastic whose resistance to degradation has contributed to widespread environmental accumulation. Enzymatic PET depolymerization has emerged as a promising bioremediation strategy, but PET hydrolysis remains challenging due to the insoluble and semi-crystalline nature of PET and the poor thermostability of many PET hydrolases at elevated temperatures. Here, several electrostatically supercharged PET binding modules (PBM) were fused to a PET-hydrolyzing Cutinase Catalytic Domain (CD) from the thermophilic microbe Thermobifida fusca to investigate how engineered PBM surface charge influences PET hydrolysis behavior. All PBM designs were derived from a native T. fusca family-2a carbohydrate binding module (CBM) as starting template. Since PET exhibited a substantially negative zeta potential, and accordingly, all positively supercharged PBMs displayed the strongest PET binding interactions in pull-down binding assays. However, stronger PET binding did not translate to improved hydrolysis activity for the fusion constructs. Instead, a slightly negatively charged PBM-CD fusion (D2 construct) exhibited activity comparable to the Cutinase CD on finely milled PET powder while showing substantially improved activity on intact PET discs, suggesting potential advantages for depolymerization of minimally processed PET feedstocks. Thermostability analysis identified an approximately 10 {degrees}C increase in melting temperature for the D2 fusion construct, corresponding to enhanced catalytic persistence and a shifted optimal hydrolysis temperature. Consequently, this construct exhibited an approximately 2-fold increase in long-term hydrolysis activity on milled PET and up to a 10-fold increase on intact PET discs, even at high solids loadings, compared to the native Cutinase CD. Collectively, these findings demonstrate that thermostability, rather than adsorption to PET alone, is a dominant factor governing functional persistence of PET hydrolases.

bioengineering↗

bioRxiv2025

There is an I in team: individual improvements in supercharged cellulase cocktail facilitates cooperative cellulose degradation

Lignocellulosic biomass is a vastly abundant renewable carbon source for biofuel production but its conversion to fermentable sugars is significantly hindered by an inherent recalcitrance to enzymatic degradation. Pretreatment technologies are successful in alleviating some challenges related to substrate recalcitrance, yet enzymes like cellulases still exhibit poor activity on highly crystalline and insoluble cellulose. Both cellulose and lignin present several issues with productive enzyme binding and efficient catalytic turnover. To address these bottlenecks, we employed protein supercharging to rationally design a glycosyl hydrolase (GH) family-6 exocellulase (Cel6B) and its native fused family-2a carbohydrate binding module (CBM2a) from the thermophilic cellulolytic microbe Thermobifida fusca. A total of 16 supercharged variants were designed across both GH/CBM domains and a chimeric library of 32 constructs, including the native enzyme, were synthesized and expressed in E. coli. The entire library of supercharged enzymes was tested for activity on several cellulosic substrates to identify one key construct, D5 CBM2a - WT Cel6B, that had a positively supercharged CBM2a that showed 2-3-fold higher activity on all substrates tested at pH 5.5. Purified enzyme assays confirmed that exocellulases behave quite different from their endocellulase counterparts when supercharged using similar protocols. Still, the purified D5 CBM2a - WT Cel6B mutant showed a 2.3-fold improvement in specific activity compared to native enzyme on crystalline cellulose. Analysis of melt curves depict that, while all other constructs tested have one distinct melt peak near the expected CBM melting point, domain melting is decoupled for the D5 CBM2a mutant. This effect reveals an intrinsic melting temperature of the Cel6B CD nearly 18 {degrees}C higher than the coupled melting temperature of the full-length enzyme. This unexpected catalytic domain stabilization effect of supercharged CBM2a domain is likely the driving force for activity improvements seen for this exocellulase that is otherwise prone to stalling and denaturation on the cellulose surface during processive catalytic turnover cycles. When combining this supercharged exocellulase construct with its endocellulase counterpart, our results show that supercharged enzymes that show the highest activity alone, produced the best synergistic partners. This study highlights another successful implementation of protein supercharging strategy for cellulases and provides another key piece towards building an effective synergistic cellulase cocktail for lignocellulosic biomass deconstruction.

bioengineering↗

bioRxiv2025

Reaction kinetics of procainamide dye derivatization of N-linked glycans to enable robust process analytical workflows for glycoprotein-based biologics manufacturing

N-glycosylation is a post-translational modification of proteins that represents a critical quality attribute (CQA) for therapeutics like monoclonal antibodies (mAbs), directly affecting drug efficacy, safety, and stability. Real-time CQA monitoring analytical platforms depend on rapid N-glycan release and fluorophore labeling chemistries to support automated bioprocess analytics during mAb manufacturing. Procainamide is a well-known fluorophore used for released N-glycans reducing sugar aldehydes labeling that offers both high fluorescence and mass spectrometry detection sensitivity comparable to several commercial reagents available in the market. However, currently there are no studies that optimize its use and long incubation times are often reported in the literature for procainamide labeling of N-glycans that has limited its use in time-sensitive workflows relevant to various stakeholders in industry, academia, and regulatory agencies. Here, we have systematically determined the combined procainamide labeling via reductive amination/reduction reaction kinetics at various incubation intervals, ranging from 1 min to 12 h, using N-glycans isolated from model biologic glycoprotein trastuzumab (TmAb). Labeling efficiencies were quantified using high-performance liquid chromatography with fluorescence detection (HPLC-FLR), and detailed reaction parameters were determined by fitting suitable kinetic models. Results indicate that most N-glycans reached over 95% labeling efficiency within 1 hour at the desired reaction temperature. Interestingly, N-glycan structural features, particularly galactosylation and fucosylation levels, significantly influenced the labeling reaction rate. Fucosylated glycans exhibited up to 4-fold higher reaction rate constants than non-fucosylated forms, whereas increased galactosylation levels was associated with slower reaction rate. These results provide essential kinetic benchmarks for incorporating procainamide labeling for released N-glycans, and facilitating more efficient analytical workflows for Process Analytical Technology (PAT) focused on biologics N-glycan analysis in both research and industrial settings.

biochemistry↗

bioRxiv2024

Time-resolved tracking of cellulose biosynthesis and microfibril network assembly during cell wall regeneration in live Arabidopsis protoplasts

Significance StatementCellulose is a major extracellular matrix component of cells that is critical for plant development and has applications to bioenergy, agricultural food/feed, textile, and wood production. Cellulose is thought to be assembled by the closely coordinated motion of plasma membrane-embedded cellulose synthase enzyme complexes. To date, however, it has not been possible to visualize de novo plant cell wall synthesis at the single cell level with the necessary spatiotemporal resolution to derive a data-driven model of how plant cells can resynthesize and assemble cell wall after its removal. Based on our time-resolved data, we propose a new model for cellulose biosynthesis after successfully performing live protoplast time-lapse imaging to visualize for the first time the complex dynamics of de novo cellulose biosynthesis and assembly into an intertwined microfibril network. Plant cell walls are composed of polysaccharides among which cellulose is the most abundant component. Cellulose is processively synthesized as bundles of linear {beta}-1,4-glucan homopolymer chains via the coordinated action of multiple enzymes in cellulose synthase complexes (CSCs) embedded within the plasma cell membrane. Plant cell walls are composed of multiple layers of cellulose fibrils that form highly intertwined extracellular matrix networks. However, it is not yet clear as to how cellulose fibrils synthesized by multiple CSCs are assembled into the intricate cellulose network deposited on plant cell surfaces. Herein, we have established an in vivo time-resolved imaging platform for visualizing cellulose during its biosynthesis and assembly into a complex fibrillar network on the surface of Arabidopsis thaliana mesophyll protoplasts as the primary cell wall regenerates. We performed total internal reflection fluorescence microscopy (TIRFM) with fluorophore-conjugated tandem carbohydrate binding modules (tdCBMs) that were engineered to specifically bind to nascent cellulose fibrils. Together with a well-controlled environment, it was possible to monitor in vivo cellulose fibril synthesis dynamics in a time-resolved manner for nearly one day of continuous cell wall regeneration on protoplast cell surfaces. Our observations provide the basis for a novel model of cellulose fibril network development in protoplasts driven by complex interplay of multi-scale dynamics that include: rapid diffusion and coalescence of short nascently synthesized cellulose fibrils; processive elongation of single fibrils; and cellulose fibrillar network rearrangement during cell wall maturation. This platform is valuable for exploring mechanistic aspects of cell wall synthesis while visualizing cellulose microfibrils assembly.

plant biology↗

bioRxiv2024

Cell-free expression and biochemical characterization of polysaccharide-synthesizing glycosyltransferases

Polysaccharides are a major class of natural polymers found abundantly across all major life forms and play a critical role as structural, metabolic, or functional components in biomolecular processes. Some polysaccharides like cellulose and hyaluronan are synthesized by membrane-bound family-2 glycosyltransferases (GTs). Despite the fact that the GT-2 family has the maximum number of deposited sequences, the biochemistry of GT-2 family enzymes is still poorly understood due to difficulties associated with GT membrane protein expression, purification, and reconstitution in lipid carriers. Here, we chose Populus tremula x tremuloides cellulose synthase 8 (PttCesA8) and Streptococcus equisimilis hyaluronan synthase (SeHas) as putative family-2-GTs to be expressed in a wheat-germ-based cell-free expression (CFE) system as proteoliposomes. The cell-free products were obtained as reconstituted liposomes directly from CFE reactions at high yields and short processing times compared to other approaches. GT enzymes expression was confirmed using SDS-PAGE and immunoblotting, and the integration of GTs in lipid layers was observed using cryogenic electron microscopy. Both GTs tested were catalytically active when incubated with their respective substrates and cofactors. The Michalis-Menten kinetic constants, Km for PttCesA8, was 295.8 {micro}M, and SeHas was 321.51 {micro}M (toward UDP N-Acetyl Glucosamine) and 207.88 {micro}M (toward UDP Glucuronic Acid), respectively. UDP was found to actively inhibit both these GTs with apparent inhibition constants of 10.08 {micro}M and 24.38 {micro}M. Mutation of specific conserved residues in structure-deficit SeHas confirmed the importance of lysine-139, glutamine-248, and threonine-283 residues in hyaluronan biosynthesis. In summary, wheat-germ-based CFE can be used to express functionally active and liposome-reconstituted family-2 GTs at high yields with relative ease to enable classical enzymology assays and will also enable more detailed structural studies in the near future.

bioengineering↗

bioRxiv2023

Characterizing the Cellulose Binding Interactions of Type-A Carbohydrate-Binding Modules using Acoustic Force Spectroscopy

A critical understanding of carbohydrate binding modules (CBMs) is vital for the manipulation of a variety of biological functions they support, including biomass deconstruction, polysaccharide biosynthesis, pathogen defence, and plant development. The unbinding characteristics of CBMs from a polysaccharide substrate surface can be studied using rupture force measurements since it enables a quantitative inference of binding properties through the application of dynamic force spectroscopy (DFS) theory. With the increase in usage of CBMs for diverse applications, it is important to engineer and characterize CBMs that have desired sets of interactions with various carbohydrate substrates. However, though the effect of mutations in the binding motif residues is known to influence CBM binding affinity, its effect on the rupture forces is still not well quantified. This is primarily due to the low experimental throughput of most single-molecule DFS techniques available to characterize the force-induced dissociation of protein-ligand interactions. Here, we have determined the rupture forces of microscopic beads functionalized with various wild-type and mutant CBMs using a highly multiplexed DFS technique called Acoustic Force Spectroscopy (AFS). We have characterized the acoustic force-induced dissociation of specifically two type A CBMs (i.e., CBM3a and CBM64) and relevant seven binding motif targeting CBM mutants unbinding from a nanocellulose surface, over a broad range of DFS loading rates (i.e., 0.1 pN/s to 100 pN/s). Our analysis of the rupture force DFS data yields apparent CBM-cellulose bond interaction parameters, which enables a quantitative comparison of the effect of corresponding mutations on cellulose-CBM binding interactions that compares favorably with results from classical bulk ensemble based binding assays. In summary, detailed insights into the rupturing mechanism of multi-CBM fused domains provides motivation for usage of specific constructs for industrial biotechnological applications.

biophysics↗

bioRxiv2023

Supercharging carbohydrate-binding module alone enhances endocellulase thermostability, binding, and activity on cellulosic biomass

Lignocellulosic biomass recalcitrance to enzymatic degradation necessitates high enzyme loadings incurring large processing costs for industrial-scale biofuels or biochemicals production. Manipulating surface charge interactions to minimize non-productive interactions between cellulolytic enzymes and plant cell wall components (e.g., lignin or cellulose) via protein supercharging has been hypothesized to improve biomass biodegradability, but with limited demonstrated success to date. Here we characterize the effect of introducing non-natural enzyme surface mutations and net charge on cellulosic biomass hydrolysis activity by designing a library of supercharged family-5 endoglucanase Cel5A and its native family-2a carbohydrate binding module (CBM) originally belonging to an industrially relevant thermophilic microbe Thermobifida fusca. A combinatorial library of 33 mutant constructs containing different CBM and Cel5A designs spanning a net charge range of -52 to 37 was computationally designed using Rosetta macromolecular modelling software. Activity for all mutants was rapidly characterized as soluble cell lysates and promising mutants (containing mutations either on the CBM, Cel5A catalytic domain, or both CBM and Cel5A domains) were then purified and systematically characterized. Surprisingly, often endocellulases with mutations on the CBM domain alone resulted in improved activity on cellulosic biomass, with three top-performing supercharged CBM mutants exhibiting between 2-5-fold increase in activity, compared to native enzyme, on both pretreated biomass enriched in lignin (i.e., corn stover) and isolated crystalline/amorphous cellulose. Furthermore, we were able to clearly demonstrate that endocellulase net charge can be selectively fine-tuned using protein supercharging protocol for targeting distinct substrates and maximizing biocatalytic activity. Additionally, several supercharged CBM containing endocellulases exhibited a 5-10 {degrees}C increase in optimal hydrolysis temperature, compared to native enzyme, which enabled further increase in hydrolytic yield at higher operational reaction temperatures. This study demonstrates the first successful implementation of enzyme supercharging of cellulolytic enzymes to increase hydrolytic activity towards complex lignocellulosic biomass derived substrates.

bioengineering↗

bioRxiv2023

Temporal Effects of Galactose and Manganese Supplementation on Monoclonal Antibody N-Linked Glycosylation in Fed-Batch and Perfusion Bioreactor Operation

Monoclonal antibodies (mAbs) represent a majority of biotherapeutics on the market today. These glycoproteins undergo post-translational modifications, such as N-linked glycosylation, that influence the structural & functional characteristics of the antibody. Glycosylation is a heterogenous post-translational modification that may influence therapeutic glycoprotein stability and clinical efficacy, which is why it is often considered a critical quality attribute (CQA) of the mAb product. While much is known about the glycosylation pathways of Chinese Hamster Ovary (CHO) cells and how cell culture chemical modifiers may influence the N-glycosylation profile of the final product, this knowledge is often based on the final cumulative glycan profile at the end of the batch process. Building a temporal understanding of N-glycosylation and how mAb glycoform composition responds to real-time changes in the biomanufacturing process will help build integrated process models that may allow for glycosylation control to produce a more homogenous product. Here, we look at the effect of specific nutrient feed media additives (e.g., galactose, manganese) and feeding times on the N-glycosylation pathway to modulate N-glycosylation of a Herceptin biosimilar mAb (i.e., Trastuzumab). We deploy the N-GLYcanyzer process analytical technology (PAT) to monitor glycoforms in near real-time for bench-scale bioprocesses operated in both fed-batch and perfusion modes to build an understanding of how temporal changes in mAb N-glycosylation are dependent on specific media additives. We find that Trastuzumab terminal galactosylation is sensitive to media feeding times and intracellular nucleotide sugar pools. Temporal analysis reveals an increased desirable production of single and double galactose-occupied glycoforms over time under glucose-starved fed-batch cultures. Comparable galactosylation profiles were also observed between fed-batch (nutrient-limited) and perfusion (non-nutrient-limited) bioprocess conditions. In summary, our results demonstrate the utility of real-time monitoring of mAb glycoforms and feeding critical cell culture nutrients under fed-batch and perfusion bioprocessing conditions to produce higher-quality biologics.

biochemistry↗

bioRxiv2023

Plant cellulose synthase membrane protein isolation directly from Pichia pastoris protoplasts, liposome reconstitution, and its enzymatic characterization

The most abundant renewable biopolymer on earth, viz., cellulose, acts as carbon storage reserve in plant and microbial cell walls that could potentially be converted into biofuels or other valuable bioproducts. Cellulose is synthesized by a plant cell membrane-integrated processive glycosyltransferase (GT) called cellulose synthase (CesA). Since only a few of these plant CesAs have been purified and characterized to date, there are huge gaps in our mechanistic understanding of these enzymes. Furthermore, the coordination between different CesAs involved in primary and secondary cell wall formation is yet to be unveiled. The biochemistry and structural biology studies of CesAs are currently hampered by challenges associated with their expression and extraction at high yields. To aid in understanding CesA reaction mechanisms and to provide a more efficient CesA extraction method, two putative plant CesAs - PpCesA5 from Physcomitrella patens and PttCesA8 from Populus tremula x tremuloides that are involved in primary and secondary cell wall formation in plants were expressed using Pichia pastoris as an expression host. We developed a protoplast-based membrane protein extraction approach to directly isolate both these membrane-bound enzymes for purification, as detected by immunoblotting and mass spectrometry-based analyses. Our method results in a higher purified protein yield by 3-4-fold than the standard cell homogenization protocol. Our purified CesAs were reconstituted into liposomes to yield active enzymes that gave similar biochemical characteristics (e.g., substrate utilization and cofactor requirements, no primer needed to initiate polymerization reaction) as enzymes isolated using the standard protocol. This method resulted in reconstituted CesA5 and CesA8 with similar Michaelis-Menten kinetic constants, Km = 167 M, 108 M and Vmax = 7.88x10-5 mol/min, 4.31x10-5 mol/min, respectively, in concurrence with the previous studies. Taken together, these results suggest that CesAs involved in primary and secondary cell wall formation can be expressed and purified using a simple and more efficient extraction method. This could potentially help unravel the mechanism of native and engineered cellulose synthase complexes involved in plant cell wall biosynthesis.

bioengineering↗

bioRxiv2023

Site-specific Effector Protein Functionalization to Create Bead-based Avidity Model Systems

The cooperative effect of multiple affinity binding interactions creating a stable bond, known as avidity, is a universal biological phenomenon seen in diverse systems. For example, avidity based biomolecular interactions are particularly important in assessing the potency of potential drugs such as monoclonal antibodies, chimeric antigen receptor (CAR) T-cell, or Natural Killer, cells to treat cancer or engineering microbes with cell surface immobilized enzyme complexes for consolidated bioprocessing (CBP) of cellulosic biomass to fuels and chemicals. However, predicting or measuring avidity based on in vitro single affinity interactions with non-complexed protein-ligand binding model systems has limitations and often fails to describe the avidity effects observed in vivo with cell surface complexed proteins interacting with multivalent ligands at solid interfaces. Acoustic force spectroscopy (AFS) based assays have recently emerged as a reliable method for direct avidity measurements, expressed as adhesion or rupture forces, which positively correlate with in vivo avidity interactions. However, to better understand and model avidity, in particular for cell-cell interactions and to correlate it with classical binding affinity, a cell mimetic model system with controlled avidity-related properties is needed. Here, we present a method for producing such a cell mimetic model system using "effector beads" that can be used in AFS-based avidity assays or any other bead-based avidity assay. The protein of interest is heterologously expressed and biotinylated in vivo in E. coli, purified, and subsequently tethered with streptavidin coated micron-sized beads to create effector beads. Our experimental results, combined with simulations of the multivalent binding phenomena, demonstrate the dependency of bead rupture force on its receptor protein surface density and force loading rate as well as the intrinsic kinetic binding parameters of the protein-ligand system of interest. These insights provide valuable information for designing future effector bead assays and cell avidity measurements for screening and characterization purposes for diverse applications.

bioengineering↗

bioRxiv2023

Oriented Display of Cello-Oligosaccharides for Pull-down Binding Assays to Distinguish Binding Preferences of Glycan Binding Proteins

The production of biofuels from lignocellulosic biomass using carbohydrate-active enzymes like cellulases is key to sustainable energy production. Understanding the adsorption mechanism of cellulases and associated binding domain proteins down to the molecular level details will help in the rational design of improved cellulases. In nature, carbohydrate-binding modules (CBMs) from families 17 and 28 often appear in tandem appended to the C-terminus of several endocellulases. Both CBMs are known to bind to the amorphous regions of cellulose non-competitively and show similar binding affinity towards soluble cello-oligosaccharides. Based on the available crystal structures, these CBMs may display a uni-directional binding preference towards cello-oligosaccharides (based on how the oligosaccharide was bound within the CBM binding cleft). However, molecular dynamics (MD) simulations have indicated no such clear preference. Considering that most soluble oligosaccharides are not always an ideal substrate surrogate to study the binding of CBMs to the native cell wall or cell surface displayed glycans, it is critical to use alternative reagents or substrates. To experimentally assess any binding directionality of CBMs towards soluble cello-oligosaccharides, we have developed a simple solid-state depletion or pull-down binding assay. Here, we specifically orient azido-labeled carbohydrates from the reducing end to alkyne-labeled micron-sized bead surfaces, using click chemistry, to mimic insoluble cell wall surface-displayed glycans. Our results reveal that both family 17 and 28 CBMs displayed a similar binding affinity towards cellohexaose-modified beads, but not cellopentaose-modified beads, which helps rationalize previously reported crystal structure and MD data. This indicates a preferred uni-directional binding of specific CBMs and could explain their co-evolution as tandem constructs appended to endocellulases to increase amorphous cellulose substrate targeting efficiency. Overall, our proposed workflow can be easily translated to measure the affinity of glycan-binding proteins to click-chemistry based immobilized surface-displayed carbohydrates or antigens.

molecular biology↗

bioRxiv2023

Engineering and characterization of carbohydrate-binding modules to enable real-time imaging of cellulose fibrils biosynthesis in plant protoplasts

O_LICarbohydrate binding modules (CBMs) are non-catalytic domains associated with cell wall degrading carbohydrate-active enzymes (CAZymes) that are often present in nature tethered to distinct catalytic domains (CD). Fluorescently labeled CBMs have been also used to visualize the presence of specific polysaccharides present in the cell wall of plant cells and tissues. C_LIO_LIPrevious studies have provided a qualitative analysis of CBM-polysaccharide interactions, with limited characterization of optimal CBM designs for recognizing specific plant cell wall glycans. Furthermore, CBMs also have not been used to study cell wall regeneration in plant protoplasts. C_LIO_LIHere, we examine the dynamic interactions of engineered type-A CBMs (from families 3a and 64) with crystalline cellulose-I and phosphoric acid swollen cellulose (PASC). We generated tandem CBM designs to determine their binding parameters and reversibility towards cellulose-I using equilibrium binding assays. Kinetic parameters - adsorption (kon) and desorption (koff) rate constants-for CBMs towards nanocrystalline cellulose were determined using quartz crystal microbalance with dissipation (QCM-D). Our results indicate that tandem CBM3a exhibits a five-fold increased adsorption rate to cellulose compared to single CBM3a, making tandem CBM3a suitable for live-cell imaging applications. We next used engineered CBMs to visualize Arabidopsis thaliana protoplasts with regenerated cell walls using wide-field fluorescence and confocal laser scanning microscopy (CLSM). C_LIO_LIIn summary, tandem CBMs offer a novel polysaccharide labeling probe for real-time visualization of growing cellulose chains in living Arabidopsis protoplasts. C_LI

plant biology↗

bioRxiv2022

Machine learning assisted ligand binding energy prediction for in silico generated glycosyl hydrolase enzyme combinatorial mutant library

Molecular docking is a computational method used to predict the preferred binding orientation of one molecule to another when bound to each other to form an energetically stable complex. This approach has been widely used for early-stage small-molecule drug design as well as identifying suitable protein-based macromolecule residues for mutagenesis. Estimating binding free energy, based on docking interactions of protein to its ligand based on an appropriate scoring function is often critical for protein mutagenesis studies to improve the activity or alter the specificity of targeted enzymes. However, calculating docking free energy for a large number of protein mutants is computationally challenging and time-consuming. Here, we showcase an end-to-end computational workflow for predicting the binding energy of pNP-Xylose substrate docked within the substrate binding site for a large library of combinatorial mutants of an alpha-L-fucosidase (TmAfc, PDB ID-2ZWY) belonging to Thermotoga maritima glycosyl hydrolase (GH) family 29. Briefly, in silico combinatorial mutagenesis was performed for the top conserved residues in TmAfc as determined by running multiple sequence alignment against all GH29 family enzyme sequences downloaded from an in-house developed Carbohydrate-Active enZyme (CAZy) database retriever program. The binding energy was calculated through Autodock Vina with pNP-Xylose ligand docking with energy minimized TmAfc mutants, and the data was then used to train a neural network model which was also validated for model predictions using data from Autodock Vina. The current workflow can be adopted for any family of CAZymes to rapidly identify the effect of different mutations within the active site on substrate binding free energy to identify suitable targets for mutagenesis. We anticipate that this workflow could also serve as the starting point for performing more sophisticated and computationally intensive binding free energy calculations to identify targets for mutagenesis and hence optimize use of wet lab resources.

bioengineering↗

bioRxiv2022

Multivariable Graphical User Interface for Simulation of Tethered Particle Motion

The analysis of particles bound to a surface by flexible tethers can facilitate understanding of various biophysical phenomena (e.g., molecular dynamics of DNA-protein or protein-ligand binding interactions, DNA extensibility and polymer biophysics). Being able to model such systems theoretically can aid in understanding experimentally observed motions and furthermore the limitations of such models can provide insight into modeling complex systems that basic theory sometimes cannot account for. The simulation of tethered particle motion (TPM) allows for efficient analysis of complex behaviors exhibited by such systems, however this type of experiment is rarely taught in undergraduate science classes. We have developed a MATLAB simulation package intended to be used in academic contexts to concisely model and graphically represent the behavior of different tether-particle systems. We show how analysis of the simulation results can be used in biophysical research employing single molecule force spectroscopy (SMFS). Here, our simulation package is capable of modeling any given particle-tether-substrate system and allows the user to generate a parameter space with static and dynamic model components. Our simulation was successfully able to recreate generally observed experimental trends using a recently developed SMFS technique called Acoustic Force Spectroscopy (AFS). Further, the simulation was validated through consideration of the conservation of energy of the tether-bead system, trend analyses, and comparison of particle positional data from actual TPM in silico experiments conducted to simulate data with a parameter space similar to the AFS experimental setup. Overall, our TPM simulator and graphical user interface is suitable for use in an academic context and serves as a template for researchers to set up TPM simulations to mimic their specific SMFS experimental setup.

scientific communication and education↗

bioRxiv2021

An Integrated Process Analytical Platform for Automated Monitoring of Monoclonal Antibody N-linked Glycosylation

The biopharmaceutical industry is transitioning towards adoption of continuous biomanufacturing practices that are often more flexible and efficient than traditional batch processes. Regulatory agencies such as the Food and Drug Administration (FDA) are further urging use of advanced PAT to analyze the design space to increase process knowledge and enable high quality biologics production. Post-translational modification of proteins, such as N-linked glycosylation are often critical quality attributes known to affect biologics safety and efficacy hence requiring close monitoring during manufacturing. Here, we developed an online sequential-injection based PAT system, called N-GLYcanyzer, that can rapidly monitor mAb glycosylation during upstream biomanufacturing. The key innovation includes design of an integrated mAb sampling and derivation system for antibody titer and glycoform analysis in under 2 hours. The N-GLYcanyzer process includes mAb capture, deglycosylation, fluorescent glycan labeling, and glycan enrichment for direct injection and analysis on an integrated high performance liquid chromatography (HPLC) system. Different fluorescent tags and reductants were tested to maximize glycan labeling efficiency under aqueous conditions, while porous graphitized carbon (PGC) was studied for optimum glycan recovery and enrichment. We find that 2-AB labeling of glycans with 2-picoline borane as a reducing agent, using the N-GLYcanyzer workflow, gives higher glycan labeling efficiency under aqueous conditions leading to upwards of a 5-fold increase in fluorescent products intensity. Finally, we showcase how the N-GLYcanyzer platform can be implemented at/on-line to an upstream bioreactor for automated and near real-time glycosylation monitoring of a Trastuzumab biosimilar produced by Chinese Hamster Ovary (CHO) cells. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/468439v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1fbb698org.highwire.dtl.DTLVardef@90da13org.highwire.dtl.DTLVardef@1dd45e5org.highwire.dtl.DTLVardef@17fadd_HPS_FORMAT_FIGEXP M_FIG N-GLYcanyzer is an automated PAT toolkit for rapid sample processing for mAb N-linked glycans analysis to enable advanced biologics manufacturing C_FIG

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