Acoustic Force Spectroscopy Reveals Subtle Differences in Cellulose Unbinding Behavior of Carbohydrate-Binding Modules: Article No. e2117467119

Markus Hackl, Edward Contrada, Jonathan Ash, Atharv Kulkarni, Jinho Yoon, Hyeon-Yeol Cho, Ki-Bum Lee, John Yarbrough, Cesar Lopez, Sandrasegaram Gnanakaran, Shishir Chundawat

Research output: Contribution to journalArticlepeer-review

1 Scopus Citations

Abstract

Protein adsorption to solid carbohydrate interfaces is critical to many biological processes, particularly in biomass deconstruction. To engineer more-efficient enzymes for biomass deconstruction into sugars, it is necessary to characterize the complex protein-carbohydrate interfacial interactions. A carbohydrate-binding module (CBM) is often associated with microbial surface-tethered cellulosomes or secreted cellulase enzymes to enhance substrate accessibility. However, it is not well known how CBMs recognize, bind, and dissociate from polysaccharides to facilitate efficient cellulolytic activity, due to the lack of mechanistic understanding and a suitable toolkit to study CBM-substrate interactions. Our work outlines a general approach to study the unbinding behavior of CBMs from polysaccharide surfaces using a highly multiplexed single-molecule force spectroscopy assay. 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 method for uniform deposition of insoluble polysaccharides on the AFS chip surfaces are demonstrated. The rupture forces of wild-type CBM3a, and its Y67A mutant, unbinding from nanocellulose surfaces suggests distinct multimodal CBM binding conformations, with structural mechanisms further explored using molecular dynamics simulations. Applying classical dynamic force spectroscopy theory, the single-molecule unbinding rate at zero force is extrapolated and found to agree with bulk equilibrium unbinding rates estimated independently using quartz crystal microbalance with dissipation monitoring. However, our results also highlight critical limitations of applying classical theory to explain the highly multivalent binding interactions for cellulose-CBM bond rupture forces exceeding 15 pN.
Original languageAmerican English
Number of pages11
JournalProceedings of the National Academy of Sciences of the United States of America
Volume119
Issue number42
DOIs
StatePublished - 2022

NREL Publication Number

  • NREL/JA-2700-84628

Keywords

  • acoustic force spectroscopy
  • biofuels
  • carbohydrate-binding module
  • nanocellulose
  • single-molecule force spectroscopy

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