Cell-free Expressed Bacteriorhodopsin in Different Soluble Membrane Mimetics: Biophysical Properties and NMR Accessibility

Abstract

SUMMARY Selecting a suitable membrane-mimicking environment is of fundamental importance for the investigation of membrane proteins. Non-conventional surfactants, such as amphipathic polymers (amphipols) and lipid bilayer nanodiscs, have been introduced as promising environments that may overcome intrinsic disadvantages of detergent micelle systems. However, structural insights into the effects of different environments on the embedded protein are limited. Here we present a comparative study of the hepta-helical membrane protein bacteriorhodopsin in detergent micelles, amphipols and nanodiscs. Our results confirm that non-conventional environments can increase stability of functional bacteriorhodopsin, and demonstrate that well-folded heptahelical membrane proteins are in principle accessible by solution-NMR methods in amphipols and phospholipid nanodiscs. Our data distinguishes regions of bacteri-orhodopsin that mediate membrane/solvent contacts in the tested environments whereas the protein’s functional inner core remains almost unperturbed. The presented data allow comparing the investigated membrane mimetics in terms of NMR spectral quality and thermal stability required for structural studies.

Modulation of Human Mitochondrial Voltage-dependent Anion Channel 2 (hVDAC-2) Structural Stability by Cysteine-assisted Barrel-lipid Interactions

“…Phase change of the lipid milieu would impose varied curvature stress on refolded hVDAC-2. Indeed, our attempts at direct refolding of both WT and C0 in DMPC vesicles did yield sufficient amount of folded protein; such poor refolding efficiency due to absence of packing defects in homogeneous preparations of long chain lipids was reported previously (32,33).…”

“…A closer examination of barrel dimensions reveals a marginal hydrophobic mismatch between the transmembrane region of hVDAC-2 (ϳ22 Å, based on hVDAC-1 structures) and DMPC (ϳ23 Å). Although it is believed that such minor differences are well accommodated with negligible loss in free energy (32), in hVDAC-2, however, we speculate that curvature stress dramatically destabilizes the barrel structure, due to lipid deformation forces (33,34). Arguably, the change toward lowered free energy of unfolding due to hydrophobic mismatch is alleviated when proteins with a longer transmembrane segment is folded into thinner bilayers (32).…”

Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding

“…These chemical shift changes cluster specifically to the Ras-like domain with little effect on the helical domain (gray and orange coloring in Gαi1 Interacts with an Activated GPCR in Phospholipid Nanodiscs. We next studied the interaction between uniformly 2 H, 15 N-labeled Gαi1Δ31 with a thermostabilized (25,26), signaling-competent (27) variant of rat neurotensin receptor subtype 1 [HTGH4 L167R (28)], which was purified from E. coli as described previously (27,29) and then incorporated in phospholipid nanodiscs (30,31 15 N-labeled Gαi1Δ31 in the apo, GDP-, or GMP-PNP-bound forms. We recorded NMR experiments with Gαi1Δ31 samples alone and after the addition of empty nanodiscs assembled with 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids and membrane scaffold protein 1D1 (MSP1D1) and finally in complex with nanodiscs containing neurotensin-activated HTGH4 (Fig.…”

Filamin A interaction with the CXCR4 third intracellular loop regulates endocytosis and signaling of WT and WHIM-like receptors

“…23,39 The ICL3 binding motif includes 6 residues predicted to lie within the receptor transmembrane domain. 45,46 To the best of our knowledge, there is no precedent in undisrupted cells for interaction between a cytoplasmic protein and one partially embedded in the membrane. Further structural analysis is necessary to determine precisely how FLNA gains access to this region.…”