Thermal activation of the co‐chaperonins GroES and gp31 probed by mass spectrometry

Abstract

Many biological active proteins are assembled in protein complexes. Understanding the (dis)assembly of such complexes is therefore of major interest. Here we use mass spectrometry to monitor the disassembly induced by thermal activation of the heptameric co-chaperonins GroES and gp31. We use native electrospray ionization mass spectrometry (ESI-MS) on a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer to monitor the stoichiometry of the chaperonins. A thermally controlled electrospray setup was employed to analyze conformational and stoichiometric changes of the chaperonins at varying temperature. The native ESI-MS data agreed well with data obtained from fluorescence spectroscopy as the measured thermal dissociation temperatures of the complexes were in good agreement. Furthermore, we observed that thermal denaturing of GroES and gp31 proceeds via intermediate steps of all oligomeric forms, with no evidence of a transiently stable unfolded heptamer. We also evaluated the thermal dissociation of the chaperonins in the gas phase using infrared multiphoton dissociation (IRMPD) for thermal activation. Using gas-phase activation the smaller (2-4) oligomers were not detected, only down to the pentamer, whereafter the complex seemed to dissociate completely. These results demonstrate clearly that conformational changes of GroES and gp31 due to heating in solution and in the gas phase are significantly different.

Native-Like and Denatured Cytochrome c Ions Yield Cation-to-Anion Proton Transfer Reaction Products with Similar Collision Cross-Sections

“…The present implementation is most similar to those reported by Robinson and coworkers [49] and later by Heeren and coworkers [50], in which a gold-plated sample capillary was enclosed in a stainless-steel capillary sleeve. The capillary sleeve was in thermal and electrical contact with the gold-plated capillary, which was electrically biased to provide the electrospray potential and was in thermal contact with the Peltier device.…”

Collision induced unfolding of protein ions in the gas phase studied by ion mobility-mass spectrometry: The effect of ligand binding on conformational stability

“…In many cases, binding affinities determined using ESI-MS show good agreement with values obtained using other means, potentially validating MS methods for use in early-stage screening in the drug discovery process [13,14]. ESI-MS is not only suitable for the analysis of protein-ligand interactions, but has widespread utility in studying large protein-protein complexes [15,16], and has been applied to the preservation and detection of very large biomolecular assemblies, including the ribosome [17,18] and the tobacco mosaic virus (Ͼ40 MDa) [19].A key underlying issue in ESI-MS studies of ligand binding is to what extent these findings can be related to solution behavior. Given the great importance of water to the protein fold [20], it seems clear that desolvation should result in catastrophic loss of native structure, with accompanying consequences for ligand binding.…”

Native Electrospray and Electron-Capture Dissociation FTICR Mass Spectrometry for Top-Down Studies of Protein Assemblies

“…51, 52 The dissociation usually results in ejection of a few protein subunits from the assembly, providing a view of the assembly’s arrangement. 25 Interestingly, ejected subunits usually carry more charges per mass than the remainder of the assembly.…”