FCS on Proteins in Crowded Environments

The most of our knowledge about proteins is mainly gained from in-vitro measurements. However, the results obtained from in-vitro measurements are likely to be deviated from the explicit natural characteristics of the proteins in the living cell. Yet, the technical difficulty of labeling proteins inside the living cell hinders carrying out in-vivo fluorescence spectroscopic studies. An approach to get closer to the complex nature of the cellular cytoplasm is to roughly mimic it by an artificial crowded-environment. Fluorescence Correlation Spectroscopy (FCS) is utilized to study diffusion effects of fluorescently labeled molecules. The implication of FCS in studying proteins mobility in a crowded-medium is limited by several technical obstacles such as distortion of the molecule detection volume[1] and switching from Brownian diffusion regime to hindered diffusion[2] regime. A pre-characterization of the viscosity and refractive index of investigated solutions and their effects on the validity of the measurement has been carried out [3]. Further, FCS was utilized to investigate the diffusional phenomena of various proteins dissolved in solutions containing classes of crowders [4].References[1] C. B. Muller, T. Eckert, A. Loman, J. Enderlein, and W. Richtering, Dual-focus fluorescence correlation spectroscopy: a robust tool for studying molecular crowding, Soft Matter, vol. 5, pp. 13581366, 2009.[2] Phillips, R. J. (2000). A hydrodynamic model for hindered diffusion of proteins and micelles in hydrogels. Biophysical Journal, 79(6), 3350-3353.[3] Junker N. (2014). Bachelor project. RWTH Aachen.[4] Hillebrecht B. S. (2015). Bachelor project. RWTH Aachen.