Proteins adopt structural ensembles which are critical for determining their functionality. Considerable progress has been made towards characterization of native structures and interactions; however, few protein folding studies have focused on measuring non-native structures. When coupled to ion mobility spectrometry-mass spectrometry (IMS-MS), variable temperature-electrospray ionization (vT-ESI) becomes a valuable technique to parse out unique protein populations hidden under a two-state cooperative folding transition. Recently, our group has provided evidence that thermodynamic information can be extracted from these unique populations. In this work, the stability and thermochemistry of differently linked polyubiquitin chains are analyzed to provide insight on how linkage sites affect polyubiquitin thermochemistry. When K48-linked and K63-linked isomers of diubiquitin are electrosprayed under native conditions, each protein produces a narrow charge state distribution centered around the 10+ and 11+ species, respectively. At high temperatures, the charge state distributions shift to broad distributions centered around the 17+ species. A gradual increase in average charge state occurs from 30 to 96 degrees Celsius for each protein and is characteristic of a cooperative two-state unfolding process due to thermal denaturation. The stability of each protein is represented by the temperature of melting (T<sub>m</sub>), where the native and denatured charge states are present at equal intensities. The T<sub>m</sub> occurs at 71 degrees Celsius for both protein isomers. When charge state abundances as a function of temperature are extracted both for ensembles and individually, thermodynamic information can be obtained from these models using a modified Gibbs-Helmholtz equation. Ensemble protein thermodynamic measurements are corroborated by differential scanning calorimetry. Individual charge state transitions are found to have unique thermochemistry values when compared between isomers despite homogeneity in their stabilities. These analyses provide insight into distinctive native and non-native solution phase structures formed by differently linked polyubiquitin chains.

Thermodynamic insights on polyubiquitin chain structure from variable temperature: Electrospray ionization and ion mobility spectrometry mass spectrometry measurements

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