Accessibility changes within diphtheria toxin T domain when in the functional molten globule state, as determined using hydrogen/deuterium exchange measurements

Abstract

The translocation domain (T domain) of diphtheria toxin adopts a partially folded state, the so-called molten globule state, to become functional at acidic pH. We compared, using hydrogen ⁄ deuterium exchange experiments associated with MS, the structures of the T domain in its soluble folded state at neutral pH and in its functional molten globule state at acidic pH. In the native state, the a-helices TH5 and TH8 are identified as the core of the domain. Based on the high-resolution structure of the T domain, we propose that TH8 is highly protected because it is buried within the native structure. According to the same structure, TH5 is partly accessible at the surface of the T domain. We propose that its high protection is caused by the formation of dimers. Within the molten globule state, high protection is still observed within the helical hairpin TH8-TH9, which is responsible for the insertion of the T domain into the membrane. In the absence of the lipid bilayer, this hydrophobic part of the domain self-assembles, leading to the formation of oligomers. Overall, hydrogen ⁄ deuterium-exchange measurements allow the analysis of interaction contacts within small oligomers made of partially folded proteins. Such information, together with crystal structure data, are particularly valuable for using to analyze the selfassembly of proteins.

MSTools—Web based application for visualization and presentation of HXMS data

“…Therefore HXMS represents a complementary technique to NMR or protein X-ray crystallography. Its speed and possibility of automation make it a technique of choice in cases where the classical structural techniques meet their limitations [5,10]. The main weakness of HXMS, its relatively low spatial resolution, can be addressed by the use of different proteases, dissociation techniques or site directed mutagenesis [11][12][13][14][15][16][17][18][19][20].…”

Structures of multidomain proteins adsorbed on hydrophobic interaction chromatography surfaces

“…Hydrogen deuterium exchange rates have been shown to be reduced by intermolecular interactions formed in bovine insulin dimers [44], diphtheria toxin oligomers [45], and from aggregation of amyloid beta peptides [46].…”

Diphtheria toxin

“…Their protonation induces partial unfolding of the domain [103]. This leads to a highly flexible and dynamic state [44][45][46][47][104][105][106] called a molten globule. Molten globule states are frequently found along the folding pathways of proteins [107,108].…”