Specific Binding at the Cellulose Binding Module–Cellulose Interface Observed by Force Spectroscopy

Abstract

The need for effective enzymatic depolymerization of cellulose has stimulated an interest in interactions between protein and cellulose. Techniques utilized for quantitative measurements of protein-cellulose noncovalent association include microgravimetry, calorimetry, and atomic force microscopy (AFM), none of which differentiate between specific protein-cellulose binding and nonspecific adhesion. Here, we describe an AFM approach that differentiates nonspecific from specific interactions between cellulose-binding modules (CBMs) and cellulose. We demonstrate that the "mismatched" interaction between murine galectin-3, a lectin with no known affinity for cellulose, and cellulose shows molecular recognition force microscopy profiles similar to those observed during the interaction of a "matched" clostridial CBM3a with the same substrate. We also examine differences in binding probabilities and rupture profiles during CBM-cellulose binding experiments in the presence and absence of a blocking agent-a substrate specific for CBM that presumably blocks binding sites. By comparison of the behavior of the two proteins, we separate specific (i.e., blockable) and nonspecific adhesion events and show that both classes of interaction exhibit nearly identical rupture forces (45 pN at ∼0.4 nN/s). Our work provides an important caveat for the interpretation of protein-carbohydrate binding by force spectroscopy; delineation of the importance of such interactions to other classes of binding warrants further study.

Multiscale Characterization of Complex Binding Interactions of Cellulolytic Enzymes Highlights Limitations of Classical Approaches

“…Single-molecule force spectroscopy in conjunction with steered MD simulations has been employed extensively to study superior mechanical stability (51), characterize the force-unfolding behavior (52), and resolve multiple binding modes of cohesin-dockerin complexes (53). However, the application of AFM-based force spectroscopy to study CBM-cellulose binding has revealed challenges in distinguishing specific vs non-specific interactions (54). Here, we have developed a novel single-molecule optical tweezer-based bond rupture assay with piconewton (pN) force resolution and millisecond (ms) time resolution (50), to understand the heterogeneity of CBM binding behavior and provide a firm molecular basis for using a certain adsorption model to interpret classical 'pull-down' assay data.…”

Nanoscale Engineering of Designer Cellulosomes

“…Indeed, there are three key lines of evidence that strongly support the idea of scaffoldin being subjected to mechanical stress: [119] (i) Detailed inspection of the scaffoldin architecture of different natural cellulosomes reveals the presence of "connecting" cohesin modules between anchoring points of the system (CBM and SLH modules (Figures 4 and 10) that mediate binding between the bacterial cell and its cellulosic substrate) and "hanging" modules outside the anchoring points (not expected to be subject to mechanical stress). Initial SMFS studies on cohesins revealed that modules located in the "connecting" region of scaffoldin are extremely mechanically stable, much more so than those in the hanging region; [119] (ii) SMFS and Steer Molecular Dynamics simulations of the cohesin modules showed the existence of a mechanical resistance region, the so-called "mechanical clamp" motif, [119,136] (iii) The anchoring points of the system, CBM-substrate and scaffoldin-cell complexes, have extremely high affinity binding constants with the cell anchoring being covalent in at least one case.…”

Real-time single molecular study of a pretreated cellulose hydrolysis mode and individual enzyme movement

“…AFM recognition imaging and single-molecule dynamic force spectroscopy (SMDFS) were applied to map the natural and pretreated plant cell wall surface and to study the affinity between noncatalytic family 3 carbohydrate-binding modules (CBM 3a) and crystalline cellulose [ 19 ]. CBM 3a has been demonstrated to specifically interact with crystalline cellulose, and has therefore been chosen as the probe to specifically recognize and map crystalline cellulose distributions [ 20 ]. In this study, we explicitly tracked individual cellulases and key, pretreated cellulose surface properties.…”