Green Proteorhodopsin Reconstituted into Nanoscale Phospholipid Bilayers (Nanodiscs) as Photoactive Monomers

Abstract

Over 4,000 putative proteorhodopsins (PRs) have been identified throughout the oceans and seas of the Earth. The first of these eubacterial rhodopsins was discovered in 2000 and has expanded the family of microbial proton pumps to all three domains of life. With photophysical properties similar to those of bacteriorhodopsin, an archaeal proton pump, PRs are also generating interest for their potential use in various photonic applications. We perform here the first reconstitution of the minimal photoactive PR structure into nanoscale phospholipid bilayers (nanodiscs), to better understand how protein-protein and protein-lipid interactions influence the photophysical properties of PR. Spectral (steady-state and time-resolved UV-visible spectroscopy) and physical (size-exclusion chromatography and electron microscopy) characterization of these complexes confirms the preparation of a photoactive PR monomer within nanodiscs. Specifically, when embedded within a nanodisc, monomeric PR exhibits a titratable pKa (6.5–7.1) and photocycle lifetime (~100–200 ms) that is comparable to the detergent solubilized protein. These ndPRs also produce a photoactive blue-shifted absorbance, centered at 377 or 416 nm, that indicates protein-protein interactions from a PR oligomer are required for a fast photocycle. Moreover, we demonstrate how these model membrane systems allow modulation of the PR photocycle by variation of the discoidal diameter (i.e., 10 or 12 nm), bilayer thickness (i.e., 23 or 26.5 Å), and degree of saturation of the lipid acyl chain. Nanodiscs also offer a highly stable environment of relevance to potential device applications.

The light‐driven sodium ion pump: A new player in rhodopsin research

“…3A and B). While it has been reported that oligomerization affects the protein stability and kinetics of the photocycle [41,42], a number of biochemical and biophysical studies have proved that the functional unit of ion-pumping rhodopsins is the monomer [43][44][45], and the physiological role of oligomerizations is still unclear. Further studies are needed to determine why ion pumping rhodopsins form oligomers under physiological conditions.…”

Functionally Active Membrane Proteins Incorporated in Mesostructured Silica Films

“…21–26 However, preparing synthetic hosts that contain membrane proteins with native or native-like activities is difficult, given the highly specific conditions required to support the generally complex protein structures 27,28 and dynamics 29,30 that are necessary for protein function. In most cases, membrane proteins can remain functional outside of the native lipid membranes only in the presence of membrane-mimetic surfactants or lipids 31–35 and over narrow ranges of solvent compositions and temperatures. 36 Such stability considerations have limited the incorporation of membrane proteins into only a few types of synthetic materials, including hydrogels, 37 block copolymers, 38–40 silica gels or glasses, 41,42 self-assembled lipid bilayer arrays, 43,44 and supported lipid bilayers.…”

Physicochemical Factors Controlling the Activity and Energy Coupling of an Ionic Strength-gated ATP-binding Cassette (ABC) Transporter

“…In the last decade, the lipid bilayer nanodisc system developed by the Sligar laboratory has become an important tool to characterize membrane proteins (19 -20). Many proteins have been reconstituted in nanodiscs, including human tissue factor (21), bacteriorhodopsin (22)(23), G-protein-coupled receptors (24 -26), chemoreceptor Tar (27)(28), translocon SecYEG (29 -31), and F 0 F 1 -ATPase (32). ABC transporters, such as the maltose system MalEFGK (33-34), the lipid A exporter MsbA (35), and P-glycoprotein (19,36), have also been reconstituted in nanodiscs, albeit generally with a low coupling efficiency.…”