The Use of Amphipols for NMR Structural Characterization of 7-TM Proteins

Abstract

While amphipols have been proven useful for refolding of seven transmembrane helical (7-TM) proteins including G-protein coupled receptors (GPCRs) and it could be shown that an amphipol environment is in principle suitable for NMR structural studies of the embedded protein, high-resolution NMR insights into amphipol refolded and isotopically labelled GPCRs are still very limited. Here we report on recent progress towards NMR structural studies of the melanocortin-2 and -4 receptor, two class A GPCRs which so far have not been reported to be incorporated into an amphipol environment. Making use of the established 7-TM protein bacteriorhodopsin (BR) we initially tested and optimized amphipol refolding conditions. Most promising conditions were transferred to the refolding of the two melanocortin receptors. Analytical scale refolding experiments on the melanocortin-2 receptor show very similar behavior to results obtained on BR. Using cell-free protein expression we could generate sufficient amounts of isotopically labeled bacteriorhodopsin as well as melanocortin-2 and -4 receptors for an initial NMR analysis. Upscaling of the amphipol refolding protocol to protein amounts needed for NMR structural studies was, however, not straight forward and impeded detailed NMR insights for the two GPCRs. While well resolved and dispersed NMR spectra could only be obtained for bacteriorhodopsin, a comparison of NMR data recorded on the melanocortin-4 receptor in SDS and in an amphipol environment indicates that amphipol refolding induces larger structural modifications in the receptor.

Amphipols for Each Season

“…For refolding from SDS, the most usual procedure is to precipitate the dodecylsulfate as its potassium salt in the presence of APols (Bazzacco et al 2012; Catoire et al 2010a; Dahmane et al 2009, 2013; Pocanschi et al 2006). Systematic studies using BR as a model have shown that other approaches can be made to work, such as dilution or dialysis, the precipitation method, however, providing the best yields (Dahmane et al 2013; Elter et al 2014). …”

“…Solution NMR spectra of BR either solubilized in DDM, trapped by A8-35, or inserted into NDs indicate that the transmembrane region of the protein is essentially the same in the three environments, but that there are some differences in the structure and/or dynamics of the extramembrane loops (Etzkorn et al 2013). The NMR spectra of A8-35-trapped BR are of a sufficient quality to expect that, given proper labeling, it should be possible to collect high-resolution data on the structure and dynamics of APol-trapped GPCRs (Elter et al 2014; Etzkorn et al 2013). Preliminary data show that the extramembrane loops of the major outer membrane protein (MOMP) from Chlamydia trachomatis trapped in A8-35 are amenable to a solution NMR study (Feinstein et al 2014).…”

Folding and stability of integral membrane proteins in amphipols

“…A case in point is solution NMR, whose conditions tend 1065 to be harsh (reviewed in Refs. [60,91,158,159] [64], which may be related to stabilization. In vaccination, better 1072 protection against a bacterial disease is obtained following immu-1073 nization against an APol-trapped than a detergent-solubilized 1074 outer TMP used as immunogen, which may be related either to 1075 its stabilization and/or to a better presentation to the immune 1076 system [53,55,161].…”

Long-Term Stability of a Vaccine Formulated with the Amphipol-Trapped Major Outer Membrane Protein from Chlamydia trachomatis

“…Elter et al 2014; Planchard et al 2014). As of today, the approach has been applied to five MPs, three β -barrel ones, OmpX (Bazzacco et al 2012; Catoire et al 2010b, 2009; Etzkorn et al 2014) and the transmembrane domains of OmpA from E. coli (Dahmane et al 2011; Zoonens et al 2005) and from Klebsiella pneumoniae (Planchard et al 2014; Renault 2008), and two α-helical ones, BR (Etzkorn et al 2013, 2014; Raschle et al 2010) and the LTB2 leukotriene receptor (Catoire et al 2011, 2010a).…”