From Compact to String—The Role of Secondary and Tertiary Structure in Charge-Induced Unzipping of Gas-Phase Proteins

Abstract

In the gas phase, protein ions can adopt a broad range of structures, which have been investigated extensively in the past using ion mobility-mass spectrometry (IM-MS) based methods. Compact ions with low number of charges undergo a Coulomb-driven transition to partially folded species when the charge increases and finally form extended structures with presumably little or no defined structure when the charge state is high. However, with respect to the secondary structure, IM-MS methods are essentially blind. Infrared (IR) spectroscopy, on the other hand, is sensitive to such structural details and there is increasing evidence that helices as well as β-sheet-like structures can exist in the gas phase, especially for ions in low charge states. Very recently, we showed that also the fully extended form of highly charged protein ions can adopt a distinct type of secondary structure that features a characteristic C5-type hydrogen bond pattern. Here we use a combination of IM-MS and IR spectroscopy to further investigate the influence of the initial, native conformation on the formation of these structures. Our results indicate that when intramolecular Coulomb-repulsion is large enough to overcome the stabilization energies of the genuine secondary structure, all proteins, regardless of their sequence or native conformation, form C5-type hydrogen bond structures. Furthermore, our results suggest that in highly charged proteins the positioning of charges along the sequence is only marginally influenced by the basicity of individual residues.In the gas-phase environment of a mass spectrometer, proteins can adopt a broad range of distinct structures. When electrosprayed from native, buffered solutions especially large proteins and protein complexes are known to retain features of their condensed-phase conformation -a fact that found broad application in the field of native mass spec-

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“…However, collision-induced unfolding allows to differentiate conformational ensembles from the way they change with internal energy [83]. When the total charge repulsion overcomes the intramolecular binding forces including salt bridges (akin to an intramolecular Rayleigh limit), large flexible molecules elongate to conformations much more extended than in solution [84,85]. For these reasons, intrinsically disordered proteins adopt both more compact and more extended (depending on the charge state) conformations in the gasphase compared to the solution [86 •• ].…”

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“…36 While IM-MS is sensitive to the ions' overall shape, infrared multiple photon dissociation (IRMPD) spectroscopy on IM-selected ions can provide additional details about the ions' molecular structure. 36,38 In IRMPD, ions are dissociated in a mass spectrometer in a wavelength dependent manner.…”

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“…These bands serve as spectralm arkers of the helical structure of proteins. [50][51][52] IRMPD spectroscopyw as used to determine the secondary structure of multiply protonatedu biquitin and cytochrome c (Figure 17). [52] The IRPMD spectra of both biomolecules at low and intermediate charges tates (denoted by La nd Ii n Figure 17) show the typical amide Ia nd II bands.H owever,i n the intermediate state, another amide II band appears.…”