Cold denaturation of encapsulated ubiquitin

Abstract

Theoretical considerations suggest that protein cold denaturation can potentially provide a means to explore the cooperative substructure of proteins. Protein cold denaturation is generally predicted to occur well below the freezing point of water. Here NMR spectroscopy of ubiquitin encapsulated in reverse micelles dissolved in low viscosity alkanes is used to follow cold-induced unfolding to temperatures below -25 degrees C. Comparison of cold-induced structural transitions in a variety of reverse micelle-buffer systems indicate that protein-surfactant interactions are negligible and allow the direct observation of the multistate cold-induced unfolding of the protein.

Figure 1

Summary of an intermediate state of cold-induced unfolding of encapsulated recombinant ubiquitin. The ribbon representations of the structure of encapsulated ubiquitin are color-coded according to whether a given amide N-H correlation in the ¹⁵NHSQC spectrum is less than (red) or more than (green) a given fraction of its intensity at +20 °C. Panels A, B, and C are of ubiquitin encapsulated in 70% C₄E₁₂/25% AOT/5% DTAB at −20 °C with a equilibrium water loading of 7 and employ intensity criteria of 5%, 10% and 15% respectively. Total surfactant concentration was 100 mM. The encapsulation buffer was 50 mM Na acetate, pH 5. Solutions were prepared in liquefied propane, which provided significantly narrower resonance lines than preparations in higher viscosity pentane. Panels E, F and G are of ubiquitin encapsulated in 100 mM AOT in pentane with a target water loading of 40 and at −30 °C. Panels E, F and G employ intensity criteria of 5%, 10% and 15% respectively. The buffer was 50 mM Na acetate pH 5 and 1.5 M NaCl. Spectra were collected at 750 MHz (¹H). Protein was prepared as described and further purified by HPLC. Drawn with PyMol (DeLano Scientific)