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.

Adsorption and activity of Thermomyces lanuginosus lipase on hydrophobic and hydrophilic surfaces measured with dual polarization interferometry (DPI) and confocal microscopy

“…A larger population of adsorbed TLL molecules are thought to be oriented with the active site facing the surface on hydrophobic surfaces compared to hydrophilic. A similar conclusion about proteins preferring different orientations depending on the properties on the surface has been drawn with carbonic anhydrase, whose orientation on a surface is thought to depend on the surface charge due to a strong positive domain close to the active site [37].…”

Electrostatic‐induced interfacial assembly of enzymes with nanosheets: Controlled orientation and optimized activity

“…5,6 A part of active sites might thus be buried and inaccessible, leading to a dramatic decrease in the bioactivity compared with the native enzymes. Few attempts to control the enzymatic orientation through tailoring the wetting properties 7 or surface charges 8 of the supports revealed that the rational artificial interference was not infeasible. However, in the approaches already reported, a reactive terminal tag or a protein conjugate had to be used.…”

“…However, in the approaches already reported, a reactive terminal tag or a protein conjugate had to be used. 7,8 Recently, efforts have been made toward the orientated attachment of proteins to solid surfaces through electrostatic assembly. [8][9][10] The electrostatic recognition strategy is thus believed to be a simple and biocompatible alternative to control the protein orientation.…”

“…7,8 Recently, efforts have been made toward the orientated attachment of proteins to solid surfaces through electrostatic assembly. [8][9][10] The electrostatic recognition strategy is thus believed to be a simple and biocompatible alternative to control the protein orientation. The vital element in the electrostatic-involved approach is to selectively recognize the biomolecules by the predetermined sites of solid surface.…”

Estimating Kinetic and Thermodynamic Parameters from Single Molecule Enzyme−Inhibitor Interactions

“…Recently, we demonstrated that an active enzyme, carbonic anhydrase (CA), can be controllably oriented on charged surfaces by electrostatic immobilization 9,10. The enzyme was found to form a complete layer on both the positively (terminated with a pyridinium salt) and negatively (terminated with a carboxylic acid) charged surfaces, however, with different orientations.…”