Melting Proteins: Evidence for Multiple Stable Structures upon Thermal Denaturation of Native Ubiquitin from Ion Mobility Spectrometry-Mass Spectrometry Measurements

• Published in: Journal of the American Chemical Society Vol. 139; no. 18; pp. 6306 - 6309
• Main Authors: El-Baba, Tarick J, Woodall, Daniel W, Raab, Shannon A, Fuller, Daniel R, Laganowsky, Arthur, Russell, David H, Clemmer, David E
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 10.05.2017; American Chemical Society (ACS)
• Subjects: Chemistry; Protein Conformation; Protein Denaturation; Spectrometry, Mass, Electrospray Ionization; Temperature; Ubiquitin - chemistry
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/jacs.7b02774

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 T m = 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.