title: Characterization of a CorA Mg2+ transport channel from Methanococcus jannaschii using a Thermofluor-based stability assay.
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.
authors.affiliation: University of Manchester, Manchester, UK. James.kean@postgrad.manchester.ac.uk
authors.forename: James
authors.initials: J
authors.lastname: Kean
authors.forename: Robert M
authors.initials: RM
authors.lastname: Cleverley
authors.forename: Liam
authors.initials: L
authors.lastname: O'Ryan
authors.forename: Robert C
authors.initials: RC
authors.lastname: Ford
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Prince
authors.forename: Jeremy P
authors.initials: JP
authors.lastname: Derrick
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry*; D027682:Cation Transport Proteins / Q000737:chemistry*; D002413:Cations, Divalent; D003460:Crystallization; D003902:Detergents; D006863:Hydrogen-Ion Concentration; D017017:Methanococcus / Q000737:chemistry*; D008958:Models, Molecular; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011489:Protein Denaturation; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D013696:Temperature
chemical_list: D019843:Archaeal Proteins; D027682:Cation Transport Proteins; D002413:Cations, Divalent; D003902:Detergents