Origin of Asymmetric Charge Partitioning in the Dissociation of Gas-Phase Protein Homodimers

Jurchen, John C.; Williams, Evan R.

doi:10.1021/ja0211508

Cited by 276 publications

(441 citation statements)

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“…When the monomer is sufficiently unfolded and charged, Coulombic repulsion is thought to lead to expulsion of the highly charged monomer. [30][31][32][34][35][36][37] The charge redistribution to the ejected monomer, as seen with SORI-CAD, is absent in the case of ECD. Therefore, dissociation of noncovalent bonds via ECD is thought not to alter the underlying structure of the monomer.…”

Section: Scheme 1 Reaction Pathways Of Gp31 21+ Upon Activation Usinmentioning

confidence: 99%

Electron Capture Dissociation as Structural Probe for Noncovalent Gas-Phase Protein Assemblies

et al. 2006

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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...

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Section: Scheme 1 Reaction Pathways Of Gp31 21+ Upon Activation Usinmentioning

confidence: 99%

Electron Capture Dissociation as Structural Probe for Noncovalent Gas-Phase Protein Assemblies

et al. 2006

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“…In general, a protein in an unfolded conformation may possess more available basic sites than those in tightly folded conformations. Jurchen and Williams [6] proposed that the origin of asymmetric charge partitioning in these homodimers is the result of one of the protein monomers unfolding in the dissociation transition state. …”

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confidence: 99%

“…One challenge in using mass spectrometry for the general analysis of protein complexes is understanding the dissociation mechanism of these complexes in the gas phase. Many groups [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] have reported asymmetric dissociation behaviour for large multimeric proteins. A small subunit, typically a protein monomer, is ejected from the complex during dissociation, with the monomer carrying away a disproportionate amount of charge for its relative mass.…”

mentioning

confidence: 99%

“…Using Fourier-transform mass spectrometry (FTMS), Jurchen and Williams [6] have conducted a number of studies in order to understand the origin of these charge distributions. In these experiments, isolated charge states of cytochrome c dimer were dissociated by sustained off-resonance irradiation collisionally activated dissociation (SORI-CAD).…”

mentioning

confidence: 99%

“…In these experiments, isolated charge states of cytochrome c dimer were dissociated by sustained off-resonance irradiation collisionally activated dissociation (SORI-CAD). According to Jurchen and Williams [6], the asymmetric charge distribution depends upon charge state, dissociation energy, and conformational flexibility. These studies showed that higher charge states lead to symmetric charge products while lower charge states lead to an asymmetric charge dissociation pathway.…”

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Theoretical investigations of the dissociation of charged protein complexes in the gas phase

Wanasundara

Thachuk

2007

J. Am. Soc. Mass Spectrom.

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A series of calculations, varying from simple electrostatic to more detailed semi-empirical based molecular dynamics ones, were carried out on charged gas phase ions of the cytochrome c dimer. The energetics of differing charge states, charge partitionings, and charge configurations were examined in both the low and high charge regimes. As well, preliminary free energy calculations of dissociation barriers are presented. It is shown that one must always consider distributions of charge configurations, once protein relaxation effects are taken into account, and that no single configuration dominates. All these results also indicate that in the high charge limit, the dissociation of protein complex ions is governed by electrostatic repulsion from the net charges, the consequences of which are enumerated and discussed. There are two main trends deriving from this, namely that charges will move so as to approximately maintain constant surface charge density, and that the lowest barrier to dissociation is the one that produces fragment ions with equal charges. In particular, it is shown that the charge-to-mass ratio of a fragment ion is not the key physical parameter in predicting dissociation products. In fact, from the perspective of the division of total charge, many dissociation pathways reported to be "asymmetric" in the literature should be more properly labelled as "symmetric" or "near-symmetric". The Coulomb repulsion model assumes that the timescale for charge transfer is faster than that for protein structural changes, which in turn is faster than that for complex dissociation. One challenge in using mass spectrometry for the general analysis of protein complexes is understanding the dissociation mechanism of these complexes in the gas phase. Many groups [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] have reported asymmetric dissociation behaviour for large multimeric proteins. A small subunit, typically a protein monomer, is ejected from the complex during dissociation, with the monomer carrying away a disproportionate amount of charge for its relative mass. Understanding the cause of this phenomenon will help to improve our understanding of the structural properties of noncovalent complexes.It is important to investigate how these dissociations occur and the factors that influence them. Using Fourier-transform mass spectrometry (FTMS), Jurchen and Williams [6] have conducted a number of studies in order to understand the origin of these charge distributions. In these experiments, isolated charge states of cytochrome c dimer were dissociated by sustained off-resonance irradiation collisionally activated dissociation (SORI-CAD). According to Jurchen and Williams [6], the asymmetric charge distribution depends upon charge state, dissociation energy, and conformational flexibility. These studies showed that higher charge states lead to symmetric charge products while lower charge states lead to an asymmetric charge dissociation pathway. Further, their results with different excitation energies show ...

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Direct Characterization of Protein Complexes by Electrospray Ionization Mass Spectrometry and Ion Mobility Analysis

Loo

Kaddis

2006

Mass Spectrometry of Protein Interactions

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Origin of Asymmetric Charge Partitioning in the Dissociation of Gas-Phase Protein Homodimers

Cited by 276 publications

References 56 publications

Electron Capture Dissociation as Structural Probe for Noncovalent Gas-Phase Protein Assemblies

Electron Capture Dissociation as Structural Probe for Noncovalent Gas-Phase Protein Assemblies

Theoretical investigations of the dissociation of charged protein complexes in the gas phase

Direct Characterization of Protein Complexes by Electrospray Ionization Mass Spectrometry and Ion Mobility Analysis

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