Proteins adopt the most thermodynamically stable structures under specific solution conditions, including the solvent composition and temperature. In protein folding, the predicted transient and low-abundance structures have hardly been observed directly due to limitations of detection techniques. Recently, ion mobility spectrometry (IMS) and mass spectrometry (MS) techniques were coupled with a variable temperature (vT)-electrospray ionization (ESI) source to study protein structures. Through such techniques, our group reported evidence for 41 unique solution conformations in the thermal denaturation of chymotrypsin inhibitor 2 (CI-2) in water, previously characterized as a two-state cooperative transition. Here, to further explore how solvents and temperatures affect the population of protein conformations, we study CI-2 thermal denaturation in 60:40 water: methanol by vT-ESI-IMS-MS. The same set of conformers reported in water is detected in the water: methanol mixture. The relative free energy values (ΔG) plots resembling free energy landscapes are compared in two solvents at multiple temperatures. Thermodynamic values (ΔH, ΔS and ΔC<sub>p</sub>) and their temperature dependences in the two solvents demonstrate the solvent effects on multiple conformers in CI-2 thermal denaturation. It was proposed in the recent study that large decreases in enthalpies and entropies reported in water at elevated temperatures are due to the decreased dielectric constant and the more dynamic structure of the heated solvent - water. With methanol addition to the solvent, we also observe ΔH and ΔS decreases compared to water at low temperatures, supporting our previous explanation. Detailed comparison allows rare insights into how solvents and temperatures regulate the populations of many solution structures of CI-2.

Solvent and temperature regulation of chymotrypsin inhibitor 2 conformers measured by ESI-IMS-MS

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