Surface Attachment Enhances the Thermodynamic Stability of Protein L

Surface–protein interactions exhibit rich and complex biophysics, which were probed by measuring the stability of protein L when attached to a hydroxy‐coated monolayer. Protein stabilization was observed due to surface‐induced excluded volume effects that restrict the conformational entropy of the unfolded state, and macromolecular crowding effects on the surface that are markedly different than those in solution.

Abstract

Despite the importance of protein–surface interactions in both biology and biotechnology, our understanding of their origins is limited due to a paucity of experimental studies of the thermodynamics behind such interactions. In response, we have characterized the extent to which interaction with a chemically well‐defined macroscopic surface alters the stability of protein L. To do so, we site‐specifically attached a redox‐reporter‐modified protein variant to a hydroxy‐terminated monolayer on a gold surface and then used electrochemistry to monitor its guanidine denaturation and determine its folding free energy. Comparison with the free energy seen in solution indicates that interaction with this surface stabilizes the protein by 6 kJ mol⁻¹, a value that is in good agreement with theoretical estimates of the entropic consequences of surface‐induced excluded volume effects, thus suggesting that chemically specific interactions with this surface (e.g., electrostatic interactions) are limited in magnitude.

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