Application of Multiple Length Crosslinkers to the Characterization of Gaseous Protein Structure

The speed, sensitivity, and tolerance of heterogeneity of native mass spectrometry, as well as the kinetic trapping of solution-like states during electrospray, makes mass spectrometry an attractive method to study protein structure. Increasing resolution of ion mobility measurements and mass resolving power and range are leading to the increase of the information content of intact protein measurements, and an expanded role of mass spectrometry in structural biology. Herein, a suite of different length noncovalent (sulfonate to positively charged side chain) crosslinkers was introduced via gas-phase ion/ion chemistry and used to determine distance restraints of kinetically trapped gas-phase structures of native-like cytochrome c ions. Electron capture dissociation allowed for the identification of crosslinked sites. Different length linkers resulted in distinct pairs of side chains being linked, supporting the ability of gas-phase crosslinking to be structurally specific. The gas-phase lengths of the crosslinkers were determined by conformational searches and density functional theory, allowing for the interpretation of the crosslinks as distance restraints. These distance restraints were used to model gas-phase structures with molecular dynamics simulations, revealing a mixture of structures with similar overall shape/size but distinct features, thereby illustrating the kinetic trapping of multiple native-like solution structures in the gas phase.