Binding and Movement of Individual Cel7A Cellobiohydrolases on Crystalline Cellulose Surfaces Revealed by Single-molecule Fluorescence Imaging

The efficient catalytic conversion of biomass to bioenergy would meet a large portion of energy requirements in the near future. A crucial step in this process is the enzyme-catalyzed hydrolysis of cellulose to glucose that is then converted into fuel such as ethanol by fermentation. Here we use single-molecule fluorescence imaging to directly monitor the movement of individual Cel7A cellobiohydrolases from Trichoderma reesei ( Tr Cel7A) on the surface of insoluble cellulose fibrils to elucidate molecular level details of cellulase activity. The motion of multiple, individual Tr Cel7A cellobiohydrolases was simultaneously recorded with ∼15-nm spatial resolution. Time-resolved localization microscopy provides insights on the activity of Tr Cel7A on cellulose and informs on nonproductive binding and diffusion. We measured single-molecule residency time distributions of Tr Cel7A bound to cellulose both in the presence of and absence of cellobiose the major product and a potent inhibitor of Cel7A activity. Combining these results with a kinetic model of Tr Cel7A binding provides microscopic insight into interactions between Tr Cel7A and the cellulose substrate. Background: Molecular level mechanisms underlying cellulose hydrolysis by cellulases remain poorly understood. Results: The majority of cellobiohydrolase molecules on cellulose surfaces were stationary. Conclusion: There is a need for improved understanding of cellulose properties resulting in large fractions of stalled cellulases. Significance: Dynamic single-molecule imaging of cellulases provides insights on productive/nonproductive binding and surface diffusion on cellulose.