Impact of Crowding Environment on the Stability, Conformation and Kinetics of CRABP I

Abstract

The importance of macromolecules paves the way towards a detailed molecular level investigation as all most all cellular processes occurring at the interior of cells in the form of proteins, enzymes, nd other biological molecules are significantly affected because of their crowding. Thus, exploring the role of crowding environment on the conformation, stability and denaturation/renaturation kinetics of protein molecules is of great importance. Here, CRABP I (cellular retinoic acid binding protein I) is employed as a model protein along with different molecular weights of polyethylene glycol (PEG 400, PEG 1000, PEG 2000, PEG 4000, PEG 6000, and PEG 8000) as molecular crowders. The experimental evaluations are done by accessing the protein secondary structure analysis using circular dichroism (CD) spectroscopy and unfolding/refolding kinetics using intrinsic fluorescence of CRABP I at 37 °C to mimic the in vivo crowding environment. Experimental results show that both conformation and stability of the native state of the protein is not significantly affected by the presence of crowding agents in the solution, whereas the crowding environment has a great impact on the unfolding/refolding kinetics of CRABP I. However, our findings show that not only the type of crowder but also the crowder size played a key role in the effects of excluded volume. In case of lower molecular weight of PEG (MW 400), even at 200 g/L concentration only the viscosity effect is observed whereas, for higher molecular weight of PEG (MW 1000), along with viscosity effect, excluded volume effect is noticed and, even at more higher concentration (200 g/L) of PEG 1000, excluded volume predominates over the viscosity effect. Using the transition state theory, we were also able to determine the free energies of activation for the unfolding and refolding studies from their respective rate constants. Furthermore, m, Cm and ΔG° of CRABP I are affected by crowding via urea-induced unfolding with increase in the size of PEG. Kinetic and stability outcomes presented the importance of crowding environment on the stability, conformation and unfolding/refolding kinetics of CRABP I.