Mimicking Physiological Cellular Environment of Protein Using Macromolecular Crowding Approach

The intracellular environment is highly crowded because of the presence of high concentration of biomolecules such as proteins, DNA, RNA, cytoskeleton etc and the concentration of these biomolecules in the cytoplasm is in the range of 80-400 mg ml-1 which accounts for nearly 5-40 % of the total cellular volume. These biomolecules generate a crowded medium having restricted amount of free water. Moreover, the volume occupied by co-solute is unavailable to other molecules, which results in the decrease of available volume to these molecules. Thus, macromolecular crowding can affect any reaction, which depends upon the available volume. The thermodynamic consequences of macromolecular crowding are known as excluded volume effects. Le Chatelier's principle leads to the conclusion that volume exclusion will always favor the folded state of the protein because this form is more compact and occupies lesser space than the denatured state. The volume exclusion phenomenon increases the entropy, which leads to decrease in the free energy of the system that leads to stabilization. Excluded volume shifts the equilibrium towards the native state because the free energy of the denatured states gets raised in crowded condition. It is also said that there is a direct effect of macromolecular crowding on the unfolded state because it increases the free energy of the denatured state and increases the relative stability of the folded state. Thus, macromolecular crowding forces the denatured state to become more compact. Thus it is expected that the behavior of any protein present in crowded environment is different from its behavior in the dilute solution. Thus, macromolecular crowding has made appreciable impact on protein structure, protein function, protein stability, protein folding, binding of ligand to protein, protein-protein interaction, molten globule formation and protein aggregation. Volume exclusion because of crowding agent increases the stability of protein and higher the volume exclusion greater is the stabilizing effect on protein. On the other hand, chemical interaction between crowder and protein overcomes the excluded volume effect leading to destabilization of the protein. Thus, the effect of crowder depends on the concentration, shape, size and nature of crowding agent employed.