Effect of Dextran on Protein Stability and Conformation Attributed to Macromolecular Crowding

Abstract

Thermally induced transition curves of hen egg-white lysozyme were measured in the presence of several concentrations of dextran at pH 2.0 by near-UV and far-UV CD. The transition curves were fitted to a two-state model by a non-linear, least-squares method to obtain the transition temperature ( T m), enthalpy change (Δ H u( T m)), and free energy change (Δ G u( T)) of the unfolding transition. An increase in T m and almost constant Δ H u( T m) values were observed in the presence of added dextran at concentrations exceeding ca 100 g l −1. In addition, dextran-induced conformational changes of fully unfolded protein were investigated by CD spectroscopy. Addition of high concentrations of dextran to solutions of acid-unfolded cytochrome c at pH 2.0 results in a shift of the CD spectrum from that characteristic of the fully unfolded polypeptide to that characteristic of the more compact, salt-induced molten globule state, a result suggesting that the molten globule-like state is stabilized relative to the fully unfolded form in crowded environments. Both observations are in qualitative accord with predictions of a previously proposed model for the effect of intermolecular excluded volume (macromolecular crowding) on protein stability and conformation.