Solid NMR and Biophysical Investigations of Ubiquitin Adsorbed to Mesoporous Silica

Protein interactions with solid surfaces are fundamental to various contemporary research areas such as the design of biosensors, biocompatible implants and microarrays of immobilized proteins (protein-on-a-chip assays)1. Fundamental understanding of the factors governing protein adsorption and release from surfaces can be augmented by the use of modern tools that are able to probe on molecular level the interaction of the protein with surface moieties at this heterogeneous interface and any subsequent changes to the structure of the protein2. We use solid state NMR spectroscopy as a tool to characterize, in molecular detail, the interface of proteins and solid surfaces in concert with more macroscopic in nature biophysical adsorption studies. The interaction of ubiquitin (Ubq) with mesoporous silica (MCM41) surfaces under aqueous conditions was investigated through adsorption isotherm measurements, isothermal titration calorimetry and solid-state NMR studies. We find that ubiquitin preferentially adsorbs in a reversible fashion to MCM41 under acidic conditions. We also show that the affinity of the protein to surface modified MCM41 using covalent attachment of 3-aminopropyltriethoxysilane and octyltriethoxysilane moieties to the silica surface3 diminishes Ubq affinity. Solid state NMR measurements of [U-15N]Ubq on MCM41 show intermediate linewidths narrower than the one measured for lyophilized free protein but still wider than the typical linewidth observed for crystalline Ubq samples4. Ramifications of these results as well as further NMR measurements currently underway on this system are discussed.