Gas Phase characterization of the non-covalent quaternary structure of cholera toxin and the cholera toxin B subunit pentamer
Jonathan Paul Williams 1*, Daniel Smith 1, Brian Green 2, Brian Marsden 3, Keith Jennings 4, Lynne Roberts 4 and James Scrivens 4
1 University of Warwick
2 Waters Uk, Manchester
3 oxford university
4 warwick university
* To whom correspondence should be addressed. E-mail: j.p.williams{at}warwick.ac.uk.
Submitted on October 20, 2005
Revised on November 17, 2005
Accepted on 19 January 2006
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Abstract |
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Cholera Toxin (CTx) is an AB5 cytotonic protein that has medical relevance in cholera and as a novel mucosal adjuvant. Here, we report an analysis of the non covalent homopentameric complex of cholera toxin B-chain (CTx-B5) using electrospray ionisation (ESI) triple quadrupole mass spectrometry (MS) and tandem mass spectrometry (MS/MS) and the analysis of the non covalent hexameric holotoxin (CTx-AB5) using ESI-TOFMS over a range of pH values that correlate with those encountered by this toxin following cellular uptake. We show that non covalent interactions within the toxin assemblies were maintained under both acidic and neutral conditions in the gas phase. However, unlike the related E. coli Shiga-like toxin B5 pentamer (SLTx B), the CTx B5 pentamer was stable at low pH indicating that additional interactions must be present within the latter. Structural comparison of the CTx B monomer interface reveals an additional alpha helix that is absent in the SLTx B monomer. In silico energy calculations support interactions between this helix and the adjacent monomer. These data provide insight into the apparent stabilisation of CTx B relative to SLTx B.
Key Words:
cholera toxin, mass spectrometry, noncovalent interaction, protein quaternary structure, tandem mass spectrometry
