Optimizing nanodiscs and bicelles for solution NMR studies of two β-barrel membrane proteins

Abstract

Solution NMR spectroscopy has become a robust method to determine structures and explore the dynamics of integral membrane proteins. The vast majority of previous studies on membrane proteins by solution NMR have been conducted in lipid micelles. Contrary to the lipids that form a lipid bilayer in biological membranes, micellar lipids typically contain only a single hydrocarbon chain or two chains that are too short to form a bilayer. Therefore, there is a need to explore alternative more bilayer-like media to mimic the natural environment of membrane proteins. Lipid bicelles and lipid nanodiscs have emerged as two alternative membrane mimetics that are compatible with solution NMR spectroscopy. Here, we have conducted a comprehensive comparison of the physical and spectroscopic behavior of two outer membrane proteins from Pseudomonas aeruginosa, OprG and OprH, in lipid micelles, bicelles, and nanodiscs of five different sizes. Bicelles stabilized with a fraction of negatively charged lipids yielded spectra of almost comparable quality as in the best micellar solutions and the secondary structures were found to be almost indistinguishable in the two environments. Of the five nanodiscs tested, nanodiscs assembled from MSP1D1ΔH5 performed the best with both proteins in terms of sample stability and spectral resolution. Even in these optimal nanodiscs some broad signals from the membrane embedded barrel were severely overlapped with sharp signals from the flexible loops making their assignments difficult. A mutant OprH that had two of the flexible loops truncated yielded very promising spectra for further structural and dynamical analysis in MSP1D1ΔH5 nanodiscs.

From Nanodiscs to Isotropic Bicelles: A Procedure for Solution Nuclear Magnetic Resonance Studies of Detergent-Sensitive Integral Membrane Proteins

“…The difference in the detergent:lipid ratios required to result in optimal spectra for the different MPs suggests that individual lipid affinities of IMPs dictates the presence of lipid in the final complexes. Usually, the role of lipids is probed by comparing different membrane mimetic environments to ascertain the most native-like in which to proceed with functionally relevant MP studies (Etzkorn et al, 2013; Kucharska et al, 2015). We expect our method to, in the first instance, ease these types of labor intensive investigations and in the second, to contribute to studies into the lipid specificity of individual IMPs.…”

Biophysical and biochemical studies of the outer membrane proteins OprG and OprH from Pseudomonas aeruginosa

“…An interesting intermediate case is the E. coli protein OmpW, which showed concerted hinge motions of partially β-structured loops as measured by solution NMR in 30-Fos micelles [130]. We have recently made good progress with reconstituting OprG into different kinds of lipid nanodiscs that proved suitable for further NMR studies on this protein in a more bilayer-like and hence more physiological environment [131].…”

“…His6-tagged proteins were purified from inclusion bodies and refolded following the published protocol [131,139]. Protein samples with a Strep-tag II used for NMR studies were solubilized from inclusion bodies in denaturation buffer (8 M urea, 10 mM MES pH 6.0, 0.1 mM EDTA) and loaded onto a hiTrap CM sepharose FF column (GE Healthcare Life Sciences).…”

“…As mentioned, OprH is up-regulated and overexpressed so that it becomes a major component of the OM when P. aeruginosa is grown under limiting concentrations of divalent cations. Therefore, OprH is believed to act as a surrogate for aeruginosa strain PAO1 and all OprH mutants were expressed in BL21 (DE3) E. coli cells as described in [92,131]. All OprH constructs used were without the N-terminal signal sequence (residues 1-22 were replaced with Met-1 so that Ala-23 becomes Ala-2 in our numbering system) and either with a C-terminal His6-tag or Strep-tag II (SAWSHPQFEK).…”

“…Proteins at concentrations of ~0.5 mg/ml with good purity were obtained using this procedure, which effectively removed all free arginine used in the refolding buffer. The proteins were quantitatively refolded as seen from the shift of their apparent molecular masses on SDS-PAGE gels.OprG from P. aeruginosa strain PAO1 was expressed in BL21 (DE3) E. coli cells as described in [131,139]. The OprG construct used was without the N-terminal signal sequence (residues 1 to 26 were replaced with Met1 so that His27 becomes His2 in our 65 numbering system) and with a C-terminal Strep-tag II (SAWSHPQFEK).…”