Many channels, enzymes and transporters are membrane proteins. To be functional, often they must associate to oligomers. Due to the nature of insertion in the lipid bilayers, studying membrane proteins is difficult and complicated. Conventionally, targets are synthesized in a host-cell system and reconstituted in detergents or vesicles. However, this can lead to not only inaccurate measurements but also dysfunctional products. The purpose of this work is to develop an in vitro methodology in exploring the oligomerization of membrane proteins. Nanolipoprotein particles (NLPs) are produced from mixing apolipoproteins and bicelles, followed by the removal of detergents. Results from fluorescence resonance energy transfer (FRET) and dynamic light scattering, respectively, demonstrate that bicelles can remove lipids from NLPs. This process occurs slowly, compared to typical micelle reaction rates. Two Cys-engineered bacteriorhodopsin fragments qualitatively and quantitatively validate that protomer association is achievable in a cell-free protein synthesis system with NLPs as monomer holders and bicelles as oligomer promoters. Fragment recombination is measured by cross-linking of disulfide bonds. Monomers of the smallest gap junction protein, connexin 26 (Cx26), can be expressed and embedded in NLPs. Although Cx26 hexamers are not observed by cross-linking reactions or FRET, this may reflect the requirement of additional components or folding mechanisms in its assembly.
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