title: Electrostatic orientation of enzymes on surfaces for ligand screening probed by force spectroscopy.
abstract: In this letter, we show that electrostatic immobilization provides a simple but effective approach for the immobilization and orientation of carbonic anhydrase onto charged surfaces. The enzyme is oriented differently on oppositely charged surfaces, with the majority of active sites facing upward on a positively charged surface and downward on a negatively charged surface. An array of negatively charged microscale surface patterns within a positively charged background was prepared by microcontact printing and used as the substrate to immobilize the enzymes. This enabled the probing of the enzyme orientations on the two differently charged surface regions by force spectroscopy with the same atomic force microscopy (AFM) probe modified with a thiolated sulfonamide inhibitor. The unbinding forces between the inhibitor tip and the enzyme immobilized on the two differently charged surfaces were measured. Two control experiments, blocking of the enzyme active site with a competitive inhibitor and removal of the zinc ion from the enzyme catalytic center, were employed to distinguish between specific and nonspecific interactions and to further verify the differences in enzyme orientation. Autocorrelation analysis of the force histograms was carried out to evaluate the specific single enzyme-inhibitor interaction force.
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
authors.forename: Xiuzhu
authors.initials: X
authors.lastname: Wang
authors.forename: Dejian
authors.initials: D
authors.lastname: Zhou
authors.forename: Kumar
authors.initials: K
authors.lastname: Sinniah
authors.forename: Caroline
authors.initials: C
authors.lastname: Clarke
authors.forename: Louise
authors.initials: L
authors.lastname: Birch
authors.forename: Haitao
authors.initials: H
authors.lastname: Li
authors.forename: Trevor
authors.initials: T
authors.lastname: Rayment
authors.forename: Chris
authors.initials: C
authors.lastname: Abell
mesh_terms: D000818:Animals; D001665:Binding Sites; D024402:Carbonic Anhydrase II / Q000737:chemistry*; D002417:Cattle; D008024:Ligands; D018625:Microscopy, Atomic Force / Q000379:methods*; D055672:Static Electricity; D013499:Surface Properties
chemical_list: D008024:Ligands; D024402:Carbonic Anhydrase II