Covalently Bonded Protein Surfaces on Poly(Methyl Methacrylate): Characterization by X-ray Photoelectron Spectroscopy and Atomic Force Microscopy

Abstract

X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) have been used to characterize a poly(methyl methacrylate) (PMMA) surface with covalently attached proteins. Protein-terminated PMMA surfaces have possible applications as substrates for bio-reactive surfaces or biosensors. The PMMA surfaces were first aminated using hexamethyldiamine; the resulting –NH2 sites were reacted with the hetero-bifunctional cross-linker Sulfo-EMCS. The N-hydroxysuccinimide ester terminal and maleimide terminal groups of Sulfo-EMCS can react with surface amine groups and sulfhydryl-containing proteins respectively. A surface that has covalently bonded with such proteins is interesting, as it may be used to develop techniques to compare the mechanical properties of proteins that have similar functions, but different structures. This study characterizes Thermotoga maritima β-glucosidase 1 (TmGH1), which belongs to a family of proteins that facilitate hydrolysis of glucose-related monomers with retention of conformation. The resulting surfaces were characterized by XPS to monitor surface composition and coverage. Tapping and contact-mode AFM methods were used to image the surface and obtain force curves. Data that suggests the creation of a covalently bonded surface of TmGH1 on PMMA has been obtained, as well as force curves using dodecanethiol-terminated AFM tips that suggest specific unfolding events when a mechanical force is applied to TmGH1. These results have applications to the further investigation of protein folding and unfolding mechanisms of family GH1 proteins using AFM-related techniques.