Characterization of a CorA Mg2+ transport channel from Methanococcus jannaschii using a Thermofluor-based stability assay

Research output: Contribution to journalArticlepeer-review

  • External authors:
  • James Kean
  • Robert M. Cleverley
  • Liam O'Ryan

Abstract

The Thermofluor assay has been a valuable asset in structural genomics, providing a high-throughput method for assessing the crystallizability of proteins. The technique has been well characterized for soluble proteins but has been less extensively described for membrane proteins. Here we show the successful application of a Thermofluor-based stability assay to an ion channel, CorA from Methanococcus jannaschii. Optimization of the concentration of free detergent within the assay was important, as excessive concentrations mask the fluorescence change associated with thermal unfolding of the protein. CorA was shown to be stabilized by low pH, but relatively insensitive to salt concentration. Divalent metal cations were also capable of stabilizing the protein, in the order Co2+>Ni2+>Mn2+>Mg2+>Ca2+. Finally, removal of the oligohistidine tag was also shown to improve the thermal stability of CorA. Conclusions are drawn from this detailed study about the general applicability of this technique to other membrane proteins. © 2008 Informa UK Ltd.

Bibliographical metadata

Original languageEnglish
Pages (from-to)653-661
Number of pages8
JournalMolecular Membrane Biology
Volume25
Issue number8
DOIs
Publication statusPublished - Dec 2008