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

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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Mechanical stability of bivalent transition metal complexes analyzed by single-molecule force spectroscopy

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.11.91 ◽

2015 ◽

Vol 11 ◽

pp. 817-827 ◽

Cited By ~ 6

Author(s):

Manuel Gensler ◽

Christian Eidamshaus ◽

Maurice Taszarek ◽

Hans-Ulrich Reissig ◽

Jürgen P Rabe

Keyword(s):

Transition Metal Complexes ◽

Single Molecule ◽

Mechanical Stability ◽

Force Spectroscopy ◽

Model Systems ◽

Bond Rupture ◽

Aqueous Environment ◽

Single Molecule Force Spectroscopy ◽

Bivalent Transition Metal ◽

Rupture Behavior

Multivalent biomolecular interactions allow for a balanced interplay of mechanical stability and malleability, and nature makes widely use of it. For instance, systems of similar thermal stability may have very different rupture forces. Thus it is of paramount interest to study and understand the mechanical properties of multivalent systems through well-characterized model systems. We analyzed the rupture behavior of three different bivalent pyridine coordination complexes with Cu2+ in aqueous environment by single-molecule force spectroscopy. Those complexes share the same supramolecular interaction leading to similar thermal off-rates in the range of 0.09 and 0.36 s−1, compared to 1.7 s−1 for the monovalent complex. On the other hand, the backbones exhibit different flexibility, and we determined a broad range of rupture lengths between 0.3 and 1.1 nm, with higher most-probable rupture forces for the stiffer backbones. Interestingly, the medium-flexible connection has the highest rupture forces, whereas the ligands with highest and lowest rigidity seem to be prone to consecutive bond rupture. The presented approach allows separating bond and backbone effects in multivalent model systems.

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Dynamic single-molecule force spectroscopy: bond rupture analysis with variable spacer length

Journal of Physics Condensed Matter ◽

10.1088/0953-8984/15/18/305 ◽

2003 ◽

Vol 15 (18) ◽

pp. S1709-S1723 ◽

Cited By ~ 97

Author(s):

Claudia Friedsam ◽

Angelika K Wehle ◽

Ferdinand K hner ◽

Hermann E Gaub

Keyword(s):

Single Molecule ◽

Force Spectroscopy ◽

Bond Rupture ◽

Single Molecule Force Spectroscopy ◽

Spacer Length

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Single-molecule force spectroscopy: A facile technique for studying the interactions between biomolecules and materials interfaces

Reviews in Analytical Chemistry ◽

10.1515/revac-2020-0115 ◽

2020 ◽

Vol 39 (1) ◽

pp. 116-129

Author(s):

Li Wang ◽

Yuhong Qian ◽

Yantao Sun ◽

Bin Liu ◽

Gang Wei

Keyword(s):

Single Molecule ◽

Materials Science ◽

Force Spectroscopy ◽

Functional Materials ◽

Dynamic Force ◽

Single Molecule Force Spectroscopy ◽

Design And Synthesis ◽

Atomic Force ◽

Potential Applications ◽

Force Mapping

AbstractThe quantification of the interactions between biomolecules and materials interfaces is crucial for design and synthesis functional hybrid bionanomaterials for materials science, nanotechnology, biosensor, biomedicine, tissue engineering, and other applications. Atomic force spectroscopy (AFM)-based single-molecule force spectroscopy (SMFS) provides a direct way for measuring the binding and unbinding forces between various biomolecules (such as DNA, protein, peptide, antibody, antigen, and others) and different materials interfaces. Therefore, in this review, we summarize the advance of SMFS technique for studying the interactions between biomolecules and materials interfaces. To achieve this aim, firstly we introduce the methods for the functionalization of AFM tip and the preparation of functional materials interfaces, as well as typical operation modes of SMFS including dynamic force spectroscopy, force mapping, and force clamping. Then, typical cases of SMFS for studying the interactions of various biomolecules with materials interfaces are presented in detail. In addition, potential applications of the SMFS-based determination of the biomolecule-materials interactions for biosensors, DNA based mis-match, and calculation of binding free energies are also demonstrated and discussed. We believe this work will provide preliminary but important information for readers to understand the principles of SMFS experiments, and at the same time, inspire the utilization of SMFS technique for studying the intermolecular, intramolecular, and molecule-material interactions, which will be valuable to promote the reasonable design of biomolecule-based hybrid nanomaterials.

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Mechanically induced silyl ester cleavage under acidic conditions investigated by AFM-based single-molecule force spectroscopy in the force-ramp mode

Faraday Discussions ◽

10.1039/c3fd00119a ◽

2014 ◽

Vol 170 ◽

pp. 357-367 ◽

Cited By ~ 14

Author(s):

Sebastian W. Schmidt ◽

Michael F. Pill ◽

Alfred Kersch ◽

Hauke Clausen-Schaumann ◽

Martin K. Beyer

Keyword(s):

Bond Dissociation Energy ◽

Single Molecule ◽

Dissociation Energy ◽

Force Spectroscopy ◽

Likelihood Estimation ◽

Bond Rupture ◽

Decay Kinetics ◽

Single Molecule Force Spectroscopy ◽

Bond Dissociation ◽

Force Clamp

AFM-based dynamic single-molecule force spectroscopy was used to stretch carboxymethylated amylose (CMA) polymers, which have been covalently tethered between a silanized glass substrate and a silanized AFM tip via acid-catalyzed ester condensation at pH 2.0. Rupture forces were measured as a function of temperature and force loading rate in the force-ramp mode. The data exhibit significant statistical scattering, which is fitted with a maximum likelihood estimation (MLE) algorithm. Bond rupture is described with a Morse potential based Arrhenius kinetics model. The fit yields a bond dissociation energy De = 35 kJ mol−1 and an Arrhenius pre-factor A = 6.6 × 104 s−1. The bond dissociation energy is consistent with previous experiments under identical conditions, where the force-clamp mode was employed. However, the bi-exponential decay kinetics, which the force-clamp results unambiguously revealed, are not evident in the force-ramp data. While it is possible to fit the force-ramp data with a bi-exponential model, the fit parameters differ from the force-clamp experiments. Overall, single-molecule force spectroscopy in the force-ramp mode yields data whose information content is more limited than force-clamp data. It may, however, still be necessary and advantageous to perform force-ramp experiments. The number of successful events is often higher in the force-ramp mode, and competing reaction pathways may make force-clamp experiments impossible.

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