Phase separation of integral membrane proteins in Triton X-114 solution.

Structure of bacteriorhodopsin at 1.55 A resolution.

Interaction of membrane proteins and lipids with solubilizing detergents.

Self-assembled phospholipid bilayer Nanodiscs have become an important and versatile tool among model membrane systems to functionally reconstitute membrane proteins. Nanodiscs consist of lipid domains encased within an engineered derivative of apolipoprotein A-1 scaffold proteins, which can be tailored to yield homogeneous preparations of disks with different diameters, and with epitope tags for exploitation in various purification strategies. A critical aspect of the self-assembly of target membranes into Nanodiscs lies in the optimization of the lipid:protein ratio. Here we describe strategies for performing this optimization and provide examples for reconstituting bacteriorhodopsin as a trimer, rhodopsin, and functionally active P-glycoprotein. Together, these demonstrate the versatility of Nanodisc technology for preparing monodisperse samples of membrane proteins of wide-ranging structure.

https://depts.washington.edu/wmatkins/PDF/ReconMembrane.pdf

Chapter 11 - Reconstitution of membrane proteins in phospholipid bilayer nanodiscs.

Electrostatic calculations of the pKa values of ionizable groups in bacteriorhodopsin

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2878%2980427-X

Formation and properties of bacteriorhodopsin monomers in the non-ionic detergents octyl-β-D-glucoside and triton X-100

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2879%2980552-9

Bacteriorhodopsin monomers pump protons.

Bacteriorhodopsin (BR) molecules are arranged as immobilized trimers in the hexagonal crystalline lattice of the purple membrane. Experiments were designed to answer the question whether the hexagonal state of aggregation is required for proper functioning of this light-driven proton pump. Using the detergents Triton X-100 and octylglucoside BR was solubilized and incorporated as monomers into large unilamellar lipid vesicles. The thermotropic behaviour of these vesicles, i. e. of the lipid phase and of the protein-protein interactions, was monitored by means of the fluorescence polarization probe DPH and circular dichroism (CD) measurements, respectively. Light-induced pH-changes of vesicle suspensions were observed with a pH-electrode. The presence of exciton coupling features in the visible CD spectrum of aggregated BR and its absence in monomeric BR allow the determination of its aggregation state. In accordance with previous work1 the results indicate that at temperatures below the lipid phase transition, BR crystallizes into patches with the same hexagonal lattice as is observed in purple membrane, whereas above the transition the lattice disaggregates and the BR molecules are monomeric. In DMPC-BR vesicles the CD transition curve caused by changes in the aggregation state of BR is shifted by about 6–8°C towards lower temperatures in comparison to the lipid phase transition. The midpoint of the latter transition (about 23°C) was not changed due to the presence of BR. Illumination resulted in an alkalization of the external medium independently of the BR aggregation state.

Norbert A. Dencher

Papers

Formation and properties of bacteriorhodopsin monomers in the non-ionic detergents octyl-β-D-glucoside and triton X-100

Bacteriorhodopsin monomers pump protons.

Aggregated and Monomeric Bacteriorhodopsin Pumps Protons