ABSTRACT
In contrast to the natural environment, proteins are commonly overexpressed and then isolated for experiments in vitro in dilute buer solutions. It remains an intriguing question to what extent the natural crowding environment inuences protein function, structure, and stability. In this chapter, we analyze the impact of various cosolutes (dextran, poly[ethylene glycol] [PEG], glucose, urea, salts, and aqueous ionic liquids) on the protein stability of two model proteins: ubiquitin and RNase A. We probe the thermal stability
CONTENTS 13.1 Introduction 247 13.2 ermodynamic Characterization of Protein Folding Process by Dierential
Scanning Calorimetry 248 13.3 ermal Unfolding of Ubiquitin in Crowded Environments 249 13.4 Heat Capacity Change of Ubiquitin 250 13.5 ermodynamic Analysis of Cosolute Eect on Ubiquitin 252 13.6 Stability of RNase A in Aqueous Solutions of Ionic Liquids 253 13.7 ermodynamic Fingerprint of Protein Folding in Cosolute Solutions 256 Acknowledgments 256 References 257
of both proteins in these articial crowding environments by dierential scanning calorimetry (DSC). e dierence in free energy of unfolding of both proteins in the presence of cosolutes to the dilute solution, ΔΔGu, is then dissected into its enthalpic and entropic contributions (ΔΔHu and ΔΔSu) in order to get mechanistic insights into the stabilizing or destabilizing mechanism of each cosolute (Senske et al. 2014). Intriguingly, even though cosolutes follow oen simple size-dependent linear concentration dependencies on the free energy level, their underlying enthalpic and entropic components (ΔΔHu and ΔΔSu) can be manifold (Sukenik et al. 2013b).
