Molecular origins of reduced activity and binding commitment of processive cellulases and associated carbohydrate-binding proteins to cellulose III

Chundawat, Shishir P. S.; Nemmaru, Bhargava; Hackl, Markus; Brady, Sonia K.; Johnson, Madeline M.; Chang, Sungrok; Lang, Matthew J.; Huh, Hyun Sue; Lee, Sanghyuk; Yarbrough, John M.; López, César A.; Gnanakaran, S.

doi:10.1016/j.jbc.2021.100431

Cited by 26 publications

(44 citation statements)

References 69 publications

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“…Although there is some overlap of bondlifetimes obtained at 0.1 pN/s and 1 pN/s for both WT and Y67A, the fitted Equation 2 inadequately describes the data for both shape factors . A similar observation of bond-lifetime data not following classical models was made recently for another type-A CBM1 and its Y31A mutant using optical tweezers (29), although the force-dependent bond-lifetime was obtained in force-clamp mode. Surprisingly, no significant difference in the force-dependent bond-lifetime was found between CBM1 and its Y31A mutant for most rupture forces.…”

Section: Rupture Force Analysis and Application Of Thesupporting

confidence: 82%

“…However, a computational study of CBM1 from T. reesei revealed that CBM1 can diffuse from the hydrophilic to the hydrophobic surface of a cellulose I crystal during which multiple local energy minima with distinct orientations were sampled (50). Similarly, it has been shown that CBM1 can bind in a non-canonical orientation when binding to cellulose III (29), which further indicates that type-A CBMs potentially display a much larger range of binding orientations on crystalline cellulose surfaces. Single-molecule imaging revealed that CBM1 exhibits distinct surface binding events (22), which could be correlated to distinct regions of crystalline cellulose, and such binding modes may be found in structurally similar type-A CBMs such as CBM3a.…”

Section: Discussionmentioning

confidence: 99%

“…The unbinding rate for CBM3a WT, from sulfuric acid-derived microfibrils isolated from poplar, extracted from SMFS rupture force data using the Bell's model was estimated to be 0.0089 s -1 (28) and is close to the value obtained in our study. However, both the Bell and DHS models assume a one-dimensional unbinding pathway, which may not represent the underlying molecular interactions based on the multimodal rupture force distributions measured in this study, as well as evidence of different CBM binding orientations to crystalline cellulose that give rise to multiple non-equivalent binding sites (50), (29).…”

Section: Rupture Force Analysis and Application Of Thementioning

confidence: 98%

“…Although AFM measures force and distance with pN and nm resolution, the determination of unbinding forces occurs far from equilibrium due to the relatively high loading rates inherent to the AFM instrument, potentially obscuring multimodal unbinding behavior seen at physiologically relevant conditions. Alternatively, optical tweezers (OT) have been used to study CBM unbinding at lower loading rates and force clamp mode (29), and the results suggest a more complex unbinding behavior where the bond-lifetime data does not follow a single exponential decay function as suggested by AFM studies (28).…”

Section: Main Text Introductionmentioning

confidence: 99%

“…Most commonly, streptavidin-coated beads are used to connect biotinylated DNA tethers (32) due to high specificity and binding strength (33,34). On the other end of the tether, the protein of interest is either covalently linked through a thiol-maleimide crosslink (23) or tethered noncovalently via the histidine tag to Anti-his antibodies, which in turn are covalently linked to aminated DNA tethers (29). The histidine tag of proteins was used previously to non-covalently link to DNA (35)(36)(37)(38) and directly attaching proteins to NTA-modified AFM tips (39).…”

Section: Main Text Introductionmentioning

confidence: 99%

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Acoustic Force Spectroscopy Reveals Subtle Differences in Cellulose Unbinding Behavior of Carbohydrate-Binding Modules

Hackl

Contrada

Ash

et al. 2021

Preprint

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To rationally engineer more efficient cellulolytic enzymes for cellulosic biomass deconstruction into sugars for biofuels production, it is necessary to better understand the complex enzyme-substrate interfacial interactions. Carbohydrate binding modules (CBM) are often associated with microbial surface-tethered cellulosomal or freely secreted cellulase enzymes to increase substrate accessibility. However, it is not well known how CBM recognize, bind, and dissociate from polysaccharide surfaces to facilitate efficient cellulolytic activity due to the lack of mechanistic understanding of CBM-substrate interactions. Our work outlines a general approach to methodically study the unbinding behavior of CBMs from model polysaccharide surfaces using single-molecule force spectroscopy. Here, we apply acoustic force spectroscopy (AFS) to probe a Clostridium thermocellum cellulosomal scaffoldin protein (CBM3a) and measure its dissociation from nanocellulose surfaces at physiologically relevant, low force loading rates. An automated microfluidic setup and methodology for uniform deposition of insoluble polysaccharides on the AFS chip surfaces is demonstrated. The rupture forces of wild-type CBM3a, and its Y67A mutant, unbinding from nanocellulose surface suggests distinct CBM binding conformations that can also explain the improved cellulolytic activity of cellulase tethered to CBM. Applying established dynamic force spectroscopy theory, the single-molecule unbinding rate at zero force is extrapolated and found to agree well with bulk equilibrium unbinding rates estimated independently using quartz crystal microbalance with dissipation monitoring. However, our results highlight the limitations of applying classical theory to explain the highly multivalent CBM-cellulose interactions seen at higher cellulose-CBM bond rupture forces (>15pN).

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Section: Rupture Force Analysis and Application Of Thesupporting

confidence: 82%

Section: Discussionmentioning

confidence: 99%

Section: Rupture Force Analysis and Application Of Thementioning

confidence: 98%

Section: Main Text Introductionmentioning

confidence: 99%

Section: Main Text Introductionmentioning

confidence: 99%

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Acoustic Force Spectroscopy Reveals Subtle Differences in Cellulose Unbinding Behavior of Carbohydrate-Binding Modules

Hackl

Contrada

Ash

et al. 2021

Preprint

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Engineering and characterization of carbohydrate‐binding modules for imaging cellulose fibrils biosynthesis in plant protoplasts

Jayachandran

Smith

Irfan

et al. 2023

Biotech & Bioengineering

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Carbohydrate binding modules (CBMs) are noncatalytic domains that assist tethered catalytic domains in substrate targeting. CBMs have therefore been used to visualize distinct polysaccharides present in the cell wall of plant cells and tissues. However, most previous studies provide a qualitative analysis of CBM-polysaccharide interactions, with limited characterization of engineered tandem CBM designs for recognizing polysaccharides like cellulose and limited application of CBM-based probes to visualize cellulose fibrils synthesis in model plant protoplasts with regenerating cell walls. Here, we examine the dynamic interactions of engineered type-A CBMs from families 3a and 64 with crystalline cellulose-I and phosphoric acid swollen cellulose. We generated tandem CBM designs to determine various characteristic properties including binding reversibility toward cellulose-I using equilibrium binding assays. To compute the adsorption (nk on ) and desorption (k off ) rate constants of single versus tandem CBM designs toward nanocrystalline cellulose, we employed dynamic kinetic binding assays using quartz crystal microbalance with dissipation. Our results indicate that tandem CBM3a exhibited the highest adsorption rate to cellulose and displayed reversible binding to both crystalline/amorphous cellulose, unlike other CBM designs, making tandem CBM3a better suited for live plant cell wall biosynthesis imaging applications. We used several engineered CBMs to visualize Arabidopsis thaliana protoplasts with regenerated cell walls using confocal laser scanning microscopy and wide-field fluorescence microscopy. Lastly, we also demonstrated how CBMs as probe reagents can enable in situ visualization of cellulose fibrils during cell wall regeneration in Arabidopsis protoplasts.

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CAZyme Characterization and Engineering for Biofuels Applications

Nemmaru,

DeChellis,

Patil

et al. 2024

Handbook of Biorefinery Research and Technology: Biomass Logistics to Saccharification

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Molecular origins of reduced activity and binding commitment of processive cellulases and associated carbohydrate-binding proteins to cellulose III

Cited by 26 publications

References 69 publications

Acoustic Force Spectroscopy Reveals Subtle Differences in Cellulose Unbinding Behavior of Carbohydrate-Binding Modules

Acoustic Force Spectroscopy Reveals Subtle Differences in Cellulose Unbinding Behavior of Carbohydrate-Binding Modules

Engineering and characterization of carbohydrate‐binding modules for imaging cellulose fibrils biosynthesis in plant protoplasts

CAZyme Characterization and Engineering for Biofuels Applications

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