Understanding the Biophysics of Protein-Surface Interactions

Abstract

Despite the biological and biotechnological importance of protein-surface interactions, our understanding of how and why they occur is still limited: What are the thermodynamic consequences of the interaction of proteins with surfaces? Why do proteins generally remain folded and functional on biological surfaces and cell membranes, but often unfold, adhere to, and inactivate on artificial surfaces? To respond to these questions we need to understand the biophysical origins of protein-surface interactions, however the absence of experimental methods to measure the thermodynamics of such interactions has so far precluded quantitative analysis. In response, we have developed an approach to measure the free energy of protein-surface interactions, which we have employed to explore the extent to which attachment to a specific, macroscopic surface alters the thermodynamic stability of protein L. We have achieved so by modifying protein L with the redox reporter methylene blue and then covalently tethering its N-terminus to a gold electrode passivated with a hydroxyl-terminated alkanethiol monolayer. Denaturant-induced unfolding of the surface-attached protein alters the ease with which the methylene blue reporter transfers electrons to the surface of the electrode, and therefore electrochemical techniques, such as square-wave voltammetry, allow monitoring the unfolding to extract protein stability. Comparing the thus obtained stability of the surface-attached protein to that of the same protein in bulk solution we find that surface-attachment stabilizes the protein due to excluded volume effects that restrict the conformational entropy of the unfolded state. We have also explored the role of macromolecular crowding, solvent composition and electrostatics to find that their surface biophysics are markedly different from those in bulk solution. We believe that our studies refine our understanding of the biophysics underlying protein-surface interactions, which may in turn improve the design of protein-surface pairs for protein-incorporating biotechnologies such as protein-based sensors.