Effect of Crowding Environment On Protein Kinetics and Dynamics Using Fluorescence as The Basic Technique

Abstract

Protein folding, the issue of protein stability and dynamic perturbations, is one of the fundamental importance in the living cell and the biological activities. While the polypeptide chain folds with the help of chaperone in the ribosome, the cytoplasmic component (which includes both micro-and macromolecules) influences the protein folding pathway and their dynamics. To understand their effect on the folding and dynamics, it is very much essential to monitor the effect of each component on the protein local as well as global structure and conformation. Out of the cytoplasmic components (i.e., micro and macro-molecules), we have investigated the effect of small molecules on the protein dynamics process. These small molecules in the form of inorganic salts (i.e., cations/anions: concentration of ≈150 mM) constitute 0.3 % of the total volume of the cell. For this purpose, cellular retinoic acid binding protein I (CRABP I) is used as a model protein. FCS observations revealed the impact of anions on the diffusion of protein as well as the hydrodynamic radius at room temperature. The outcome of an FCS experiment is a correlation function between intensity and time of diffusion of CRABP I in the presence of different salts. Taking a note from the impact of micromolecules, we have started observing the effects exerted by the macromolecules, such as carbohydrates (i.e., glucose, sucrose, fructose or the synthetic ones like Dextran, Ficoll and Cyclodextrin), which has an approximate concentration of ≈ 300 to 400 mg/ml in the cytosol of Escherichia coli and constitutes 20 to 40 % of the total volume, on the nature of the folding intermediates. Starting with our objective, we are trying to see how the shape and size of macromolecule bring changes in the kinetics.