Why do GroEL Ions Exhibit Two Gas Phase Conformers?

Abstract

The reasons for the existence of two gas phase conformers for electrosprayed ions of the large tetradecameric protein complex GroEL are considered. Key features are that: (1) both conformers extrapolate to very similar cross sections in the limit of zero charge; (2) both conformers supercharge above the maximum value permitted by the Rayleigh criterion; and (3) one of the conformers supercharges substantially more than the other. We hypothesize that the supercharging observed is associated to the approximately cylindrical shape of GroEL in aqueous solution, enabling accumulation of extra charge on either one or two of its bases when they intersect the evaporating drop surface. The two conformers would then correspond to cases when either one or the two bases of the cylinder carry extra charge. Apparently, the conformer symmetrically (doubly) supercharged on both ends is Coulombically stretched, therefore exhibiting a mobility different from the conformer asymmetrically supercharged only on one end. Several general consequences follow. First, non-spherical proteins may generally be charged above the usual Rayleigh limit. Second, we confirm the previously advanced but contentious notion that gas phase protein ions are readily compacted or stretched away from their crystal structure by capillary and Coulombic forces during the electrospraying process.

Ion Mobility-Mass Spectrometry Differentiates Protein Quaternary Structures Formed in Solution and in Electrospray Droplets

“…First, the origins of the bimodal IM profiles observed for both bovGDH dodecamers and SAP decamers can be linked directly to experimentally- verified structural models. While all the conformers observed for the complexes studied here likely pre-exist in solution, and previous reports have analyzed bimodal IM profiles for proteins and complexes, 13,25 no previous study has linked so clearly the relative abundances of specific protein structures to the crowding forces associated within rapidly drying nanodroplets. Furthermore, we verify our protein conformer assignments by studying key structural analogs, performing ligand binding experiments, and conducting detailed CID measurements.…”

“…Typical experiments aimed at assessing such crowding forces in vitro using model systems typically employ protein concentrations in the mM range and above. 4 While our experiments do not achieve such concentrations in bulk solution, they far exceed them during the drying steps of ESI, 13 as the probability of trapping multiple biological units within ESI droplets scales non-linearly with concentration. 3 Results shown Fig.…”

“…Recent reports, however, have pointed to ESI droplet surface charge and surface tension effects as specific mechanisms that may give rise to aberrant protein complex conformational forms, rather than aberrant protein binding stoichiometries. 13,14 …”

Structural Analysis of Activated SgrAI–DNA Oligomers Using Ion Mobility Mass Spectrometry

“…61 This is likely due to a non-globular shape of the SgrAI dimer+2PC DNA oligomers, because m/z is related to the surface area to volume ratio of the macromolecule. 61, 6263 It is possible that these were discerned only in the spectra with a higher ratio of PC DNA to SgrAI due to the formation of larger oligomers under these conditions. However, the diminished intensity in these regions with higher ratios of PC DNA to SgrAI suggests an effect by the DNA to reduce the amount of the larger oligomers, perhaps by competing with a second DNA binding site on SgrAI within the oligomer.…”

Surface induced dissociation yields substructure of Methanosarcina thermophila 20S proteasome complexes

“…It is a tetradecameric protein complex (801 kDa) consisting of two heptamer rings [11]. GroEL has served as a large protein complex model system for gas phase studies [12–15] and both ion mobility (IM)-MS and CID have been employed to study GroEL ligand binding properties [16–18]. …”