Characterization of a CorA Mg²⁺transport channel fromMethanococcus jannaschiiusing 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.

Cation permeability in CorA family of proteins

“…from three or more technical replicates of independent batches of proteoliposomes. www.nature.com/scientificreports www.nature.com/scientificreports/ (stabilization up to 95 °C) 40 . Altogether this might indicate that CoHex indeed binds to CorA proteins but either not with the high affinity or not exactly at the selectivity filter.…”

ThermoFAD, a Thermofluor®‐adapted flavin ad hoc detection system for protein folding and ligand binding

“…Thermofluor ® is an excellent candidate for this type of analysis, but suffers from the limitation that the fluorescent dyes used to determine the protein unfolding temperature interact with the detergent lipophilic moiety. This limitation makes the analysis difficult, if not impossible [20]. However, Thermo FAD allowed unfolding temperature analysis of a membrane‐anchored flavoprotein to be performed in the presence of detergents, because the flavin cofactor fluorescence is not influenced by these amphipathic molecules.…”

High-Throughput Melting-Temperature Analysis of a Monoclonal Antibody by Differential Scanning Fluorimetry in the Presence of Surfactants

“…SYPRO® Orange (Sigma–Aldrich) is known to show a bright fluorescence in presence of micelles,41 which results in a high initial fluorescence intensity. This problem was already reported in literature, especially in the field of membrane protein science 16,42–44. To overcome this problem, Kean et al43 carefully titrated the free surfactant concentration to a level with a sufficient signal‐to‐noise ratio, at which the determination of melting event was possible.…”

“…This problem was already reported in literature, especially in the field of membrane protein science. 16,[42][43][44] To overcome this problem, Kean et al 43 carefully titrated the free surfactant concentration to a level with a sufficient signal-to-noise ratio, at which the determination of melting event was possible. After this, Senisterra et al 42 demonstrated that DSLS is a suitable alternative to DSF with respect to the applicability of high-throughput thermal stability screenings in the presence of surfactants or notably hydrophobic proteins, such as membrane proteins, because no fluorescent probe is needed.…”