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Ion mobility and mass spectrometry techniques are coupled with a temperature-controlled electrospray ionization source to follow the structural transitions of ubiquitin in aqueous solution (pH = 3) at elevated solution temperatures (T = 26–96 °C). Changes in the charge state distribution are consistent with a two-state, cooperative unfolding transition having a melting temperature of Tm = 71 ± 2 °C, in agreement with prior measurements Wintrode, P. L.; Makhatadze, G. I.; Privalov, P. L. Proteins, 1994, 18, 246−253. However, analysis of ion mobility distributions reveals the two-state transition is a composite of transitions involving at least nine unique species: three native or native-like structures; two that appear to be equilibrium intermediates (i.e., populations of new conformers that form at elevated temperatures but subsequently disappear at higher temperatures); and four products observed at high temperatures, including the well-characterized ubiquitin A state, and two solution species that are differentiated based on a cis- or trans-configured Glu18-Pro19 peptide bond. These nine states vary in abundances by factors as large as ∼103 over the range of solution temperatures. Although experimental melting transitions are conceived as a loss of well-defined structure leading to a random distribution of unstructured, denatured forms, the results provide evidence for new conformers having at least some well-defined structural elements are stabilized as temperature is increased.
El‐Baba et al. (Fri,) studied this question.