Protein diffusion, stability and activity in crowded media

Molecular crowding at the cellular level is capable of changing the properties of the macromolecules involved in disparate biological processes and it is thought to affect differently small and large molecules. Here we present a study of ovalbumin and neuroserpin diffusion both in hen egg albumen and i n two systems containing ovalbumin and Ficoll70, respectively, to model media with moderate to very high viscosity. These proteins belong to a class of proteins, called serpins, characterized by a high structural homology and different functions. Particularly, neuroserpin, a serine protease inhibitor, is involved in a rare conformational genetic disease (FENIB). We used two techniques for studying different aspects of the molecular motion: Fluorescence Correlation Spectroscopy, that reveals the self-diffusion of particles labeled with a fluorescent probe, and Dynamic Light Scattering, that measures the collective diffusion of all particles, as affected by intermolecular interactions. A cysteine-containing variant of the wild type neuroserpin was produced to get a fluorescently labeled system, without affecting protease inhibition or serpin polymerization. Effects of the crowded environment on both protein diffusion and functional and dysfunctional reaction kinetics were studied.