title: Investigating receptor-ligand systems of the cellulosome with AFM-based single-molecule force spectroscopy.
abstract: Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e. optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (>120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.
authors.affiliation: Lehrstuhl für Angewandte Physik and Center for Nanoscience, Ludwig-Maximilians-Universität.
authors.forename: Markus A
authors.initials: MA
authors.lastname: Jobst
authors.forename: Constantin
authors.initials: C
authors.lastname: Schoeler
authors.forename: Klara
authors.initials: K
authors.lastname: Malinowska
authors.forename: Michael A
authors.initials: MA
authors.lastname: Nash
mesh_terms: D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism; D044603:Cellulosomes / Q000737:chemistry* / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry* / Q000378:metabolism; D055767:Immobilized Proteins / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D018625:Microscopy, Atomic Force / Q000379:methods*; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism; D000097722:Cohesins
chemical_list: D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D055767:Immobilized Proteins; D008024:Ligands; D009097:Multienzyme Complexes; D011956:Receptors, Cell Surface; D000097722:Cohesins