Hydrogen/deuterium exchange mass spectrometry and optical spectroscopy as complementary tools for studying the structure and dynamics of a membrane protein

Abstract

The hydrophobic nature of membrane proteins poses considerable challenges for the application of traditional analytical techniques. Hydrogen/deuterium exchange (HDX) with electrospray mass spectrometry (ESI-MS) detection is a potentially very powerful approach for investigating the structure and dynamics of membrane proteins, but thus far this technique has been applied mostly to soluble species. By using ESI-MS in conjunction with low temperature size exclusion chromatography (SEC) the current study explores the global HDX behavior of the integral membrane protein bacteriorhodopsin (BR) under various conditions. The experiments are complemented by UV–vis absorption measurements that report on the environment of the protein's retinal chromophore and fluorescence spectroscopy which probes the extent of resonance energy transfer between Trp residues and retinal. In agreement with previous reports, our HDX data imply that amide hydrogens in the transmembrane helices of native BR are highly protected, whereas most sites in solvent-exposed loops are readily exchangeable. Low temperature SEC/ESI-MS allows the labile Schiff-base linkage between protein and retinal to be completely preserved, such that the HDX properties of co-existing protein populations can be monitored individually. Solubilization of BR in sodium dodecyl sulfate (SDS) induces major structural changes and hydrolytic loss of retinal. The SDS-denatured protein appears to retain a significant degree of stable helical structure (around 25%, compared to the native state value of 74%). Bicelle-mediated refolding of SDS-denatured bacterioopsin in the presence of free retinal regenerates a native-like BR conformation with a yield close to 0.9. The HDX/SEC/ESI-MS approach developed in this work should be applicable to other membrane protein systems as well.