SIMPLE ASSAY FOR ADSORPTION OF PROTEINS TO THE AIR-WATER INTERFACE

A rapid assay is described, based upon the Marangoni effect, which detects the formation of a denatured-protein film at the air-water interface (AWI) of aqueous samples. This assay requires no more than a 20 microliter aliquot of sample, at a protein concentration of no more than 1 mg/ml, and it can be performed with any buffer that is used to prepare grids for electron cryo-microscopy (cryo-EM). In addition, this assay provides an easy way to estimate the rate at which a given protein forms such a film at the AWI. Use of this assay is suggested as a way to pre-screen the effect of various additives and chemical modifications that one might use to optimize the preparation of grids, although the final proof of optimization still requires further screening of grids in the electron microscope. In those cases when the assay establishes that a given protein does form a sacrificial, denatured-protein monolayer, it is suggested that subsequent optimization strategies might focus on discovering how to improve the adsorption of native proteins onto that monolayer, rather than to prevent its formation. A second alternative might be to bind such proteins to the surface of rationally designed affinity grids, in order to prevent their diffusion to, and unwanted interaction with, the AWI.

RECONSTITUTION OF A PROTEIN MONOLAYER ON THIOLATES FUNCTIONALIZED GaAs SURFACE

In the aim to realize an efficient resonant biosensor, gallium arsenide (GaAs) presents many advantages. In addition to its properties of transduction, GaAs is a crystal for which microfabrication processes were developed, conferring the possibility to miniaturize the device and integrate electronic circuit. Moreover, the biofunctionalization could be realized on the crystalline surface without layer deposition, constituting a real advantage to perform reusable sensor. The functionalization of GaAs surface was engaged in order to immobilize a protein monolayer on this substrate. Functionalization was done using a mixed self assembled monolayer of thiolate molecules. Characterizations at micro and nanoscale were performed to control the surface state, the establishment of thiolates self-assembled monolayer, the surface atomic composition and the topography of the GaAs substrate at the different steps of the process. Protein immobilization on thiolates modified GaAs was revealed through a detailed AFM study and in situ MALDI-TOF MS and MS/MS modified surface interrogations.

PROTEIN MONOLAYER FORMATION AT AIR–ELECTROLYTE INTERFACE: A LANGMUIR–BLODGETT STUDY

The interfacial surface activity of a protein, ovalbumin (OVA) at bare air/water interface in presence and also in absence of electrolyte (KCl) in subphase has been investigated. The surface activity was measured as a function of time. It has been found that, the presence of KCl in aqueous subphase enhances the adsorption rate of the protein. The changes of area/molecule, compressibility, rigidity and unfolding of OVA are trivial up to 10 mM KCl concentration. These properties of OVA, above 10 mM KCl concentration are significant and have been explained in the perspective of DLVO theory and many-body ion–protein dispersion potentials. The presence of high concentration of electrolyte increases the β-structure of OVA, resulting into larger unfolding as well as larger intermolecular aggregates. The overall study indicates that KCl perturbs the OVA monolayer.