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Nanoscale Dynamics of Cellulase TrCel7A Digesting Cellulose

Zachary K. Haviland, Daguan Nong, Kate L. Vasquez Kuntz, Thomas J. Starr, Dengbo Ma, Ming Tien, Charles T. Anderson, View ORCID ProfileWilliam O. Hancock
doi: https://doi.org/10.1101/2021.02.18.431891
Zachary K. Haviland
aDepartment of Biomedical Engineering, Pennsylvania State University, University Park, PA 16802.
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Daguan Nong
aDepartment of Biomedical Engineering, Pennsylvania State University, University Park, PA 16802.
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Kate L. Vasquez Kuntz
bDepartment of Biology, Pennsylvania State University, University Park, PA 16802.
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Thomas J. Starr
bDepartment of Biology, Pennsylvania State University, University Park, PA 16802.
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Dengbo Ma
cDepartment of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802.
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Ming Tien
cDepartment of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802.
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Charles T. Anderson
bDepartment of Biology, Pennsylvania State University, University Park, PA 16802.
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William O. Hancock
aDepartment of Biomedical Engineering, Pennsylvania State University, University Park, PA 16802.
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  • ORCID record for William O. Hancock
  • For correspondence: woh1@psu.edu
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Abstract

Understanding how cellulases catalyze the digestion of lignocellulose is a major goal of bioenergy research. Cel7A from Trichoderma reesei is a model exoglucanase that degrades cellulose strands from their reducing ends by processively cleaving individual cellobiose units. Despite being one of the most studied cellulases, the binding and hydrolysis mechanisms of Cel7A are still debated. We used single-molecule tracking to analyze the dynamics of 11,116 quantum dot-labeled TrCel7A binding to and moving processively along immobilized Gluconoacetobacter cellulose. Enzyme molecules were localized with a spatial precision of a few nanometers and followed for hundreds of seconds. Most enzymes bound into a static state and dissociated without detectable movement. Processive enzymes moved an average distance of 39 nm at an average speed of 3.2 nm/s. Static binding episodes preceding and following processive runs were of similar duration to static binding events that lacked any processive movement. Transient jumps of >20 nm were observed, but no diffusive behavior indicative of a diffusive search of the enzyme for a free cellulose strand end was observed. These data were integrated into a three-state model in which TrCel7A molecules can bind from solution into either a static or a processive state, and can reversibly switch between static and processive states before dissociating. From these results, we conclude that the rate-limiting step for cellulose degradation by Cel7A is the transition out of the static state either by dissociation from the cellulose surface or initiation of a processive run.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted February 22, 2021.
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Nanoscale Dynamics of Cellulase TrCel7A Digesting Cellulose
Zachary K. Haviland, Daguan Nong, Kate L. Vasquez Kuntz, Thomas J. Starr, Dengbo Ma, Ming Tien, Charles T. Anderson, William O. Hancock
bioRxiv 2021.02.18.431891; doi: https://doi.org/10.1101/2021.02.18.431891
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Nanoscale Dynamics of Cellulase TrCel7A Digesting Cellulose
Zachary K. Haviland, Daguan Nong, Kate L. Vasquez Kuntz, Thomas J. Starr, Dengbo Ma, Ming Tien, Charles T. Anderson, William O. Hancock
bioRxiv 2021.02.18.431891; doi: https://doi.org/10.1101/2021.02.18.431891

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