Whereas proteins generally remain stable upon interaction with biological surfaces， they frequently unfold on and adhere to artificial surfaces. Understanding the physicochemical origins of this discrepancy would facilitate development of protein-based sensors and other technologies that require surfaces that do not compromise protein structure and function. To date， however， only a small number of such artificial surfaces have been reported， and the physics of why these surfaces support functional biomolecules while others do not has not been established. Thus motivated， we have developed an electrochemical approach to determining the folding free energy of proteins site-specifically attached to chem... More
Whereas proteins generally remain stable upon interaction with biological surfaces， they frequently unfold on and adhere to artificial surfaces. Understanding the physicochemical origins of this discrepancy would facilitate development of protein-based sensors and other technologies that require surfaces that do not compromise protein structure and function. To date， however， only a small number of such artificial surfaces have been reported， and the physics of why these surfaces support functional biomolecules while others do not has not been established. Thus motivated， we have developed an electrochemical approach to determining the folding free energy of proteins site-specifically attached to chemically well-defined， macroscopic surfaces. Comparison with the folding free energies seen in bulk solution then provides a quantitative measure of the extent to which surface interactions alter protein stability. As proof-of-principle， we have characterized the FynSH3 domain site-specifically attached to a hydroxyl-coated surface. Upon guanidinium chloride denaturation， the protein unfolds in a reversible， two-state manner with a free energy within 2 kJ/mol of the value seen in bulk solution. Assuming that excluded volume effects stabilize surface-attached proteins， this observation suggests there are countervening destabilizing interactions with the surface that， under these conditions， are similar in magnitude. Our technique constitutes an unprecedented experimental tool with which to answer long-standing questions regarding the molecular-scale origins of protein-surface interactions and to facilitate rational optimization of surface biocompatibility.
