Specific binding at the cellulose binding module-cellulose interface observed by force spectroscopy.
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.
†Department of Chemistry, Duke University, Durham, North Carolina 27708, United States.
Jason R
JR
King
†Department of Chemistry, Duke University, Durham, North Carolina 27708, United States.
Carleen M
CM
Bowers
†Department of Chemistry, Duke University, Durham, North Carolina 27708, United States.|‡Department of Biochemistry, Duke University Medical School, Durham, North Carolina 27710, United States.
Eric J
EJ
Toone
D001665:Binding Sites; D002151:Calorimetry; D002482:Cellulose / Q000737:chemistry*; D018625:Microscopy, Atomic Force / Q000379:methods*; D011485:Protein Binding; D014893:Weightlessness
D002482:Cellulose