Application of Multiple Length Cross-linkers to the Characterization of Gaseous Protein Structure

Abstract

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.

Using Density Functional Theory for Testing the Robustness of Mobile-Proton Molecular Dynamics Simulations on Electrosprayed Ions: Structural Implications for Gaseous Proteins

“…The gentle nature of MPMD 300 runs prevented the ions from venturing very far from their starting points, and as a result, the ions remained trapped in native-like conformations. These gentle simulations mimic the behavior of protein ions in native ESI, explaining why the MPMD 300 results are in agreement with native ESI experiments. − ,,,− In contrast, MPMD 300–500,300 conditions allow protein ions to overcome major activation barriers, permitting them to roam across their energy landscape and settle into structures with lower free energy.…”

“…The profound structural alterations evident from points (i–iii) clash with previous data, which suggest that native ESI preserves the overall fold of ubiquitin and other proteins. − ,,,− In other words, MPMD 300–500,300 runs do not reproduce the properties of protein ions generated by native ESI. In contrast, structures that agree with available native ESI data were obtained under MPMD 300 conditions (see the preceding section, Figure B) .…”

“…Current experiments can probe some structural aspects of gaseous biomolecular ions, ,,,− but exact three-dimensional conformations or proton configurations cannot be uncovered. As a result, computational techniques play a key role for deciphering the properties of these ions.…”

“…As a result of the hydrophobic effect, proteins in solution adopt tightly packed structures where nonpolar residues are sequestered in the core, while charged and polar side chains reside on the surface where they interact favorably with water . The substantial weakening of the hydrophobic effect in the gas phase suggests that gaseous proteins might adopt “inside-out” structures, with a hydrophilic core and a hydrophobic exterior. − Yet, it appears that protein ions produced by native ESI do not adopt such inside-out conformations. ,, The survival of native-like conformations has been attributed to kinetic trapping, i.e., the presence of large activation barriers in the gas phase that prevent major structural changes on the time scale of typical ESI-IMS/MS experiments. ,,− …”

“…High computational cost makes it impractical to apply DFT to systems in the size range of proteins. However, DFT can predict conformations and proton configurations of smaller species, such as gaseous peptide ions. ,,,− For such small species, it is believed that DFT-optimized structures (i.e., those with the lowest E DFT ) accurately reflect the properties of gaseous electrosprayed ions. ,,,− The tendency of gaseous peptides to adopt relaxed low-energy structures is in contrast to protein ions after native ESI, where kinetic trapping preserves solution-like conformations that do not represent stable gas-phase structures. − ,,,− …”