Label-Free Molecular Observations of Membrane-Associated Species using Backscattering Interferometry

Membrane-associated proteins are integral components of cellular processes and disease pathogenesis. Quantitative observations of membrane protein interactions are extremely difficult; the membrane environment that is necessary to maintain appropriate structural and functional characteristics of such species interferes with or perturbs many analytical methods. In fact, typical assays to observe such interactions require the species of interest to be isolated and removed from the native membrane environment, usually with covalent modification. We will describe a label-free method to observe and evaluate membrane protein-ligand interactions in minimally-altered native membrane environments. This strategy is based on the use of backscattering interferometry (BSI) in which minute changes in the refractive index of the bulk solution caused by cognate ligand-receptor interactions are observed and quantified without the need for extrinsic molecular labeling. Combined with a method to present membrane proteins in an isotropically-scattering matrix derived from the native cellular environment, Ashlwe are able to observe a variety of cognate ligand-receptor interactions over a large range of equilibrium binding affinities.