(673h) Controlling Protein Unfolding at the Solution-Solid Interface By Modifying the Nanoscale Environment of the Surface

Many biotechnologies require proteins in their native state at solid-liquid interfaces (e.g. biocatalysis, biosensing, chromatography, biomaterials, etcâ?¦). Unfortunately proteins often adopt non-native conformations and reduced activity on surfaces. While this phenomena has been of great interest to numerous fields, and therefore the focus of many studies, the specific mechanisms of surface-mediated protein denaturation remain elusive. Traditional biophysical techniques are difficult to apply to interfacial systems due to limited signal from adsorbates relative to protein in the contacting solution. Furthermore, the information gathered from traditional approaches are difficult to interpret due to ensemble averaging of heterogeneous, dynamic systems. We studied the mechanism of lysozyme (T4L) unfolding on fused silica (FS) using single-molecule methods that provided direct insight into the cause of denaturation. The structure of T4L was monitored with FoÌ?rster resonance energy transfer (FRET) while simultaneously tracking the adsorption, diffusion, and desorption of individual molecules at the solidâ??solution interface. Single-molecule trajectories were collected for hundreds of thousands of adsorbed T4L molecules in a high throughput manner. Analysis of these trajectories suggested that the unfolding of T4L on FS was mediated by surface diffusion and occurred on isolated nanoscale sites. The frequency of these denaturing sites was controlled by changing the surface coverage, increasing protein concentration passivated denaturing sites. Since the addition of soluble factors is not always appropriate, we are investigating the effects of protein nanoscale environments in order to control the structure and diffusion of adsorbed protein. This includes nanoscale patterning of surface topography, which we propose as a method to hinder interfacial diffusion and stabilize the folded state of adsorbed protein through confinement.