Summary of an intermediate state of cold-induced unfolding of encapsulated recombinant ubiquitin. The ribbon representations of the structure of encapsulated ubiquitin13 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 described15 and further purified by HPLC. Drawn with PyMol (DeLano Scientific)