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

Abstract

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).

Determination of protein–protein interactions at the single-molecule level using optical tweezers

“…AFS can be used to study DNA-protein interactions like DNA-viral capsid protein assembly (Van Rosmalen et al, 2020), DNA-RecA filament formation, and to determine the energy landscape of digoxigenin-antidigoxigenin bonds (Sitters et al, 2014). Furthermore, AFS allows to probe the mechanical properties of cells (Sorkin et al, 2018), the strength of molecular interactions of cells with the extracellular matrix , and the unbinding behavior of carbohydrate binding modules to polysaccharide surfaces (Hackl et al, 2021). See Fig.…”

Multivariable Graphical User Interface for Simulation of Tethered Particle Motion

“…Since all these conditions are approximately valid in the considered model, Stokes Law serves as a reasonable approximation to the net effect that is observed. This also means that enough information is available such that the deviations in position within a given timestep can be extrapolated from the simulation as is outlined in Equations ( 8) through (10) below. A correction factor is introduced to account for the edge effects since the tether-bead system is near the surface throughout the simulation.…”

“…Equations ( 8) through (10) are used to update the x, y and z position at a given timestep. These cartesian position elements are then used in Eqs.…”

“…For the comparison of the simulation results with SMFS experimental data, a TPM experiment was carried out in our laboratory using an Acoustic Force Spectroscopy (AFS) instrument. 9,19 A more detailed description of the AFS experimental setup is found in the SI document and is also described in our recent publication 10 . Briefly, the surface of the AFS chip is incubated with anti-digoxigenin fab fragments for 20 minutes, followed by a surface passivation with bovine serum albumin (BSA) protein, casein protein, and Pluronic F-127 nonionic surfactant in 10mM phosphate buffered saline (PBS) solution for 30 minutes.…”

“…8 Further, several corrections are included from a series of models presented in scientific literature to increase the accuracy of the TPM simulations and allow the user to understand the limitations/uses of the calculations being made. In particular, we have generated experimental data to validate our simulation predictions using a recently developed SMFS technique called acoustic force spectroscopy (AFS) [8][9][10] . AFS is similar to optical tweezers force spectroscopy that instead uses acoustic waves to trap micron sized particles that enable SMFS measurements for diverse tethered-particle systems.…”