Influence of Crowding Agents on the Small GTPase Rab2B: Stability Consideration and Intermediate Formation.

This work investigates the thermal and chemical denaturation of recombinant Rab2B in the absence and presence of varying concentrations of denaturant (urea) under crowded environments using the synthetic crowders PEG 8 (polyethylene glycol, MW of 8 kDa) and Ficoll 70 (MW of 70 kDa). Rab2B plays an important role in vesicle trafficking, and more specifically, it is crucial in maintaining the integrity of the Golgi morphology. Our findings demonstrate that increasing concentrations of urea lead to a decrease in both the thermal and chemical stability of Rab2B in a crowder-dependent manner. A distinct formation of intermediate was induced by the crowders in the presence of 2 M urea having greater structural content as realized from CD and fluorescence studies. Furthermore, ANS fluorescence assays allowed us to monitor the changes in hydrophobicity of the protein under different conditions, while time-resolved fluorescence studies were carried out to gain information on the accessibility of the tryptophan residues. Taken in the context of the function of Rab2B, these findings contribute to a better understanding of the effect of macromolecular crowding on the stability and conformation of the protein. Since Rab2B is known to undergo a switch in conformation between GDP- and GTP-bound states and shows subsequent interaction with binding partner(s), the underlying flexibility of Rab2B and the possibility of formation of functional intermediates underscore the importance of the crowded medium in assisting the protein to execute its role in the physiological interior.