Integral Membrane Protein Fragment Recombination after Transfer from Nanolipoprotein Particles to Bicelles

Abstract

A new method for the measurement of membrane protein oligomer association is described. Two engineered fragments of bacteriorhodopsin, which are known to spontaneously associate in bicelles, were expressed in nanolipoprotein particles (NLPs or nanodiscs) using an Escherichia coli S30 cell-free synthesis system. When separately prepared NLPs containing the fragments were mixed, fragment association did not occur, indicating that the apolipoprotein edge blocks transfer between NLPs. However, when bicelles were added to this mixture, fragment association was detected by disulfide cross-linking. The rate of cross-linking was consistent with previously published equilibrium and kinetic parameters. Characterization of the NLP/bicelle mixture by dynamic light scattering and fluorescence spectroscopy indicates that the NLP bilayer transfers to bicelles in a simple reversal of the synthesis of NLPs from bicelles. These experiments validate using cell-free synthesis of membrane proteins in NLPs, followed by treatment with bicelles, as a method for measuring oligomerization of integral membrane protein subunits in a bilayer-like environment.