Stability of the homopentameric b subunits of shiga toxins 1 and 2 in solution and the gas phase as revealed by nanoelectrospray fourier transform ion cyclotron resonance mass spectrometry

Abstract

The assembly of the B subunits of Shiga toxins (Stx) 1 and 2 and the influence of solution conditions (protein concentration, temperature, pH, and ionic strength) on it are investigated using temperature-controlled nanoflow electrospray (nano-ES) ionization and Fourier-transform ion cyclotron resonance mass spectrometry. Despite the similar higher order structure predicted by X-ray crystallography analysis, the B 5 homopentamers of Stx1 and Stx2 exhibit differences in stability under the solution conditions investigated. At solution temperatures ranging from 0 to 60°C and subunit concentrations ranging from 5 to 85 M, the Stx1 B subunit exists almost entirely as the homopentamer in aqueous solutions, independent of the ionic strength. In contrast, the degree of assembly of Stx2 B subunit is strongly dependent on temperature, subunit concentration, and ionic strength. At subunit concentrations of more than 50 M, the Stx2 B subunit exists predominantly as a pentamer, although smaller multimers (dimer, trimer, and tetramer) are also evident. At lower concentrations, the Stx2 B subunit exists predominantly as monomer and dimer. The relative abundance of multimeric species of the Stx2 B subunit was insensitive to the ion source conditions, suggesting that gas-phase dissociation of the pentamer ions in the source does not influence the mass spectrum. Blackbody infrared radiative dissociation of the protonated B 5 ions of Stx2 at the ϩ12 and ϩ13 charge states proceeds, at reaction temperatures of 120 to 180°C, predominantly by the ejection of a single subunit from the complex. Dissociation into dimer and trimer ions constitutes a minor pathway. It follows that the dimer and trimer ions and, likely, the monomer ions observed in the nano-ES mass spectra of Stx2 B subunit originated in solution and not from gas-phase reactions. It is concluded that, under the solution conditions investigated, the homopentamer of Stx2 B subunit is thermodynamically less stable than that of Stx1 B subunit. Arrhenius activation parameters determined for the protonated Stx2 B 5 ions at the ϩ12 and ϩ13 charge states were compared with values reported for the corresponding B 5 ions of Stx1 B subunit. In contrast to the differential stability of the Stx1 and Stx2 B pentamers in solution, the dissociation activation energies (E a ) determined for the gaseous complexes are indistinguishable at a given charge state. The similarity in the E a values suggests that the protonated pentamer ions of both toxins are stabilized by similar intersubunit interactions in the gas phase, a result that is in agreement with the X-ray crystal structures of the holotoxins. (J Am Soc Mass Spectrom 2005, 16, 1957

Influence of coulombic repulsion on the dissociation pathways and energetics of multiprotein complexes in the gas phase

“…This is an interesting finding since it was recently shown that, in aqueous ammonium acetate solution, that Stx2 B 5 is also thermodynamically less stable than Stx1 B 5 [38]. In contrast, the Arrhenius parameters for the dissociation of the gaseous B 5 nϩ ions of Stx1 and Stx2, where n ϭ 12, 13, are indistinguishable for the same charge state [38]. These findings highlight the importance of taking into account the contribution of Coulombic repulsion when comparing the relative stabilities of structurally distinct multiprotein complexes in the gas phase.…”

“…Nevertheless, this analysis suggests that the Stx2 B 5 is significantly less stable energetically than Stx1 B 5 in the gas phase. This is an interesting finding since it was recently shown that, in aqueous ammonium acetate solution, that Stx2 B 5 is also thermodynamically less stable than Stx1 B 5 [38]. In contrast, the Arrhenius parameters for the dissociation of the gaseous B 5 nϩ ions of Stx1 and Stx2, where n ϭ 12, 13, are indistinguishable for the same charge state [38].…”

“…As described previously [38], the B subunits of Stx1 associate into a stable B 5 homopentamer in aqueous solution at neutral pH and subunit concentrations Ͼ1 M. The B 5 complex is easily transferred to the gas phase by nanoES in both positive and negative ion modes. Shown in Figure 2a …”

“…ϩn ions investigated [38], and for the lower charge states of the S 4 ϩn ions (n ϭ 14, 15) considered (Figure 8d). There are a number of possible explanations for the nonlinearity of first-order kinetic plots and these have been discussed recently [39].…”

Shiga Toxin 2 Subtypes of Enterohemorrhagic E. coli O157:H- E32511 Analyzed by RT-qPCR and Top-Down Proteomics Using MALDI-TOF-TOF-MS

“…They also noted that other reseachers had implicated the aspartic acid at residue 24 as responsible for differences in holotoxin/receptor binding [9]. The issue of Stx2 holotoxin/Gb3 receptor binding is beyond the scope of the current work; however, native state mass spectrometry [45,46], which involves analysis of higher order protein complexes by electrospray ionization mass spectrometry (ESI-MS), may be used to confirm the importance of residues 16 and 24 on holotoxin stability and Gb3 binding. In any case, MALDI-TOF-TOF-MS/MS-PSD is particularly well-suited for detecting the presence (or absence) of amino acid substitutions involving D-residues (and other residues) in a polypeptide sequence.…”