Electron capture dissociation (ECD) of proteins in Fourier transform ion cyclotron resonance mass spectrometry usually leads to charge reduction and backbone-bond cleavage, thereby mostly retaining labile, intramolecular noncovalent interactions. In this report, we evaluate ECD of the 84-kDa noncovalent heptameric gp31 complex and compare this with sustained off-resonance irradiation collisionally activated dissociation (SORI-CAD) of the same protein. Unexpectedly, the 21+ charge state of the gp31 oligomer exhibits a main ECD pathway resulting in a hexamer and monomer, disrupting labile, intermolecular noncovalent bonds and leaving the backbone intact. Unexpectedly, the charge separation over the two products is highly proportional to molecular weight. This indicates that a major charge redistribution over the subunits of the complex does not take place during ECD, in contrast to the behavior observed when using SORI-CAD. We speculate that the ejected monomer retains more of its original structure in ECD, when compared to SORI-CAD. ECD of lower charge states of gp31 does not lead to dissociation of noncovalent bonds. We hypothesize that the initial gas-phase structure of the 21+ charge state is significantly different from the lower charge states. These structural differences result in the different reaction pathways when using ECD.Since the late 1990s, electron capture dissociation 1 (ECD) has been available as a tool for structural analysis in Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). To date, ECD is routinely used for top-down sequencing and identification of the posttranslational modifications, such as sulfide bridges, phosphorylation, and methylation. [2][3][4][5][6][7] During an ECD experiment, low-energy electrons are injected into the ICR cell and captured by multiply protonated ions, resulting in the release of approximately 2-6 eV of recombination energy into the ion. 1,8 Typically, the capture of one or more electrons leads to the rapid dissociation of covalent backbone bonds, resulting in the formation of c and z‚ fragment ions. 9 The ECD process has so far been studied using peptides and proteins with a molecular mass up to 50 kDa 5,10 but not yet for protein complexes or proteins with a higher molecular mass. There is still an active debate about the exact mechanisms of ECD. 8,[11][12][13][14][15][16][17] The early research demonstrated that ECD leads to rapid dissociation of the backbone close to the site of electron capture, not necessarily fragmenting the weakest bonds in the molecule. In line with this observation, ECD has been found to often preserve labile noncovalent bonds, 10 for instance in cytochrome c 18 and vancomycin complexed with diacetyl-L-Lys-D-Ala- This makes ECD a potentially useful technique for examining interaction sites in larger, noncovalent protein complexes.In this report, we use ECD to study the interaction sites in the large gp31 heptameric protein complex from bacteriophage T4. The protein complex has a molecular mass of 84 kDa and co...
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.
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