Evidence of (anti)metamorphic properties of modified graphitic surfaces obtained in real time at a single-molecule...

Understanding protein unfolding on a surface is of vital importance in nanoscience, nanobiotechnology, and medicine. Surfaces that retain the native conformation of adsorbed proteins (antimetamorphic surfaces) represent one of the main strategies for creating biocompatible materials, which are in great demand in biotechnology. Though the influence of surfaces on protein conformation has been studied for decades, real-time investigations of protein conformational behavior on a surface obtained at the single-molecule or sub-molecular level are still lacking and remain a challenge. In this work, we apply time-lapse atomic force microscopy (AFM) in aqueous solution to visualize the conformational dynamics of individual model protein molecules (E.coli RNA polymerase, RNAP) adsorbed on modified highly oriented pyrolytic graphite (HOPG) surfaces. We quantitatively characterize the evolution of height and shape of individual RNAP molecules adsorbed on a HOPG surface modified with an oligoglycine-hydrocarbon graphite modifier (GM) during the unfolding process and determine the characteristic unfolding time as ∼7 min. Furthermore, we make a HOPG surface antimetamorphic by modifying it with a denatured RNAP protein layer. Our results provide direct evidence of GM-HOPG-induced RNAP unfolding at the single-molecule level and open new strategies for the development and investigation of antimetamorphic graphitic surfaces.