Thermal decomposition of a gaseous multiprotein complex studied by blackbody infrared radiative dissociation. Investigating the origin of the asymmetric dissociation behavior.

The blackbody infrared radiative dissociation technique was used to study the thermal decomposition of the gaseous B5 pentamer of the Shiga-like toxin I and its complexes with the Pk trisaccharide and a decavalent Pk-based oligosaccharide ligand (STARFISH, S). Dissociation of the protonated pentamer, (B5 + nH)n+ triple bond B5n+ where n = 11-14, proceeds almost exclusively by the loss of a single subunit (B) with a disproportionately large fraction (30-50%) of the parent ion charge. The degree of charge enrichment of the leaving subunit increases with increasing parent ion charge state. For n = 12-14, a distribution of product ion charge states is observed. The yields of the complementary pairs of product ions are sensitive to the reaction temperature, with higher temperatures favoring greater charge enrichment of the leaving subunit for +13 and +14, and the opposite effect for +12. These results indicate that some of the protons are rapidly exchanged between subunits in the gas phase. Dissociation of B5(14+) x S proceeds exclusively by the loss of one subunit, although the ligand increases the stability of the complex and also reduces the degree of charge enrichment in the ejected monomer. For B5(12+)(Pk)1-3, the loss of neutral Pk competes with loss of a subunit at low temperatures. Linear Arrhenius plots were obtained from the temperature-dependent dissociation rate constants measured for the loss of B from B5n+ and B514+ x S. The magnitude of the Arrhenius parameters is highly dependent on the charge state of the pentamer: Ea = 35 kcal/mol and A = 1,019 s(-1) (+14), 46 kcal/mol and 1,023 S(-1) (+13), 50 kcal/mol and 1026 s(-1) (+12), and 80 kcal/mol and 10(39) (+11). The Ea and A for B5(14+) x S are 59 kcal/mol and 10(30) s(-1), respectively. The reaction pathways leading to greater charge enrichment of the subunit lost from the B5(14+) and B5(13+) ions correspond to higher energy processes, however, these pathways are kinetically preferred at higher temperatures due to their large A factors. A simple electrostatic model, whereby charge enrichment leads to Coulombic repulsion-induced denaturation of the subunits and disruption of the intersubunit interactions, provides an explanation for the magnitude of the Arrhenius parameters and the origin of the asymmetric dissociation behavior of the complexes.

The observation of multivalent complexes of Shiga-like toxin with globotriaoside and the determination of their stoichiometry by nanoelectrospray Fourier-transform ion cyclotron resonance mass spectrometry.

We show by nanoelectrospray ionization (nanoES) Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) that it is possible to observe oligosaccharide-protein complexes with dissociation constants in the millimolar range, such as P(k) trisaccharide (globotriaoside) complexed with the Shiga-like toxin (SLT) of pathogenic E. coli. It is further demonstrated that nanoES/FT-ICR MS is an exquisite method to study quantitative aspects of the association of mono- and polyvalent oligosaccharide ligands with multimeric proteins, such as the SLTs. At increasing trisaccharide:protein ratios it was shown that the B(5 )toxin subunit complexes with 5 P(k) trisaccharides and only after all 5 copies of site 2 are essentially filled do any of the remaining 10 receptor sites become occupied. From the distribution of bound P(k)'s at the five binding sites, it was possible to establish association constants for each of the five sites and to confirm that binding occurs noncooperatively, the association constants for each site are identical and that compared to site 1, site 2 exhibits a tenfold higher affinity for the globotriaoside synthetic ligand 1. The facile identification of the occupancy of binding sites represents information that is not readily available by other techniques. This sensitive and rapid estimation of association constants for protein-ligand complexes, which are free of unpredictable secondary effects that plague enzyme linked assays, is likely to find wide application.