title: Measurements of relative binding of cohesin and dockerin mutants using an advanced ELISA technique for high-affinity interactions.
abstract: The cellulosome is a large bacterial extracellular multienzyme complex able to degrade crystalline cellulosic substrates. The complex contains catalytic and noncatalytic subunits, interconnected by high-affinity cohesin-dockerin interactions. In this chapter, we introduce an optimized method for comparative binding among different cohesins or cohesin mutants to the dockerin partner. This assay offers advantages over other methods (such as ELISA, cELIA, SPR, and ITC) for particularly high-affinity binding interactions. In this approach, the high-affinity interaction of interest occurs in the liquid phase during the equilibrated binding step, whereas the interaction with the immobilized phase is used only for detection of the unbound dockerins that remain in the solution phase. Once equilibrium conditions are reached, the change in free energy of binding (ΔΔG(binding)), as well as the affinity constant of mutants, can be estimated against the known affinity constant of the wild-type interaction. In light of the above, we propose this method as a preferred alternative for the relative quantification of high-affinity protein interactions.
authors.affiliation: Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Michal
authors.initials: M
authors.lastname: Slutzki
authors.forename: Yoav
authors.initials: Y
authors.lastname: Barak
authors.forename: Dan
authors.initials: D
authors.lastname: Reshef
authors.forename: Ora
authors.initials: O
authors.lastname: Schueler-Furman
authors.forename: Raphael
authors.initials: R
authors.lastname: Lamed
authors.forename: Edward A
authors.initials: EA
authors.lastname: Bayer
mesh_terms: D018797:Cell Cycle Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D044603:Cellulosomes / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D048013:Acetivibrio thermocellus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000097722:Cohesins
chemical_list: D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D011993:Recombinant Fusion Proteins; D000097722:Cohesins