Self-assembly of GDGTs in aqueous media. We found that the GDGTs spontaneously self-assembled into giant vesicles (1-20 µm diameter) in aqueous media upon hydration of a thin lipid film and the membranes were visualized by staining with the lipophilic dye-labeled Texas Red-DHPE (Figure , Figure ). If a water-soluble fluorescent dye such as pyranine (HPTS) was added to the hydration solution, the vesicles encapsulated the dye in the inner aqueous volume (Figure , Figure ). The GDGT vesicle membranes were observed under the microscope to display fluid-like behavior such as shape fluctuations (Figure ). However, it is noteworthy that unlike typical fluid phase phospholipids (e.g. POPC), the GDGT lipid films required increased mechanical forces (i.e. scratching, vortexing, sonication) to disperse into vesicles, suggesting that the lipids are tightly packed in the thin films. Thin films of the glucosecontaining lipids (P1Glc and PG1Glc) were harder to hydrate likely because of extensive intermolecular hydrogenbonding among the sugar headgroups. We observed that the efficiency of hydration of the lipid films depended on the nature and ionic strengths of the buffers. Tris buffer (50 mM, pH 7.5) was found to be the most suitable for hydration for all the lipids studied. Only PG2 films could readily hydrate in several other buffers like HEPES, PBS, and even in pure water to form vesicles. Also, only PG2 could hydrate to form giant vesicles in the presence of proteins such as superfolder green fluorescent protein (sfGFP) and encapsulate the protein (Figure ). Other GDGT lipids formed aggregates in the presence of protein. Next, we tested whether the multilamellar lipid dispersions can be extruded into smaller vesicles via extrusion through membrane filters. Using negative-staining transmission electron microscopy we confirmed the presence of vesicles having diameters less than 200 nm (Figure ). Given the high microviscosity of tetraether membranes and large difference in intermolecular packing as compared to typical phospholipids, it is not obvious whether integral membrane proteins of non-archaeal origin can be reconstituted into GDGT vesicles. There are only a few examples of reconstitution of non-archaeal integral membrane proteins into crude archaeal GDGT lipid membranes. Elferink et al. reconstituted bovine cytochrome c-oxidase in lipid extracts from Sulfolobus acidocaldarius primarily composed of bipolar lipids. Curiously, archaea are not known to be involved in photosynthesis, which is a heavily membrane-dependent process. Therefore, we chose to reconstitute a bacterial photosynthetic reaction center (RC) protein into GDGT membranes. First, we dissolved the protein in a mixture of octyl glucoside and PG2. Next, the detergents were removed by treatment with BioBeads followed by extensive dialysis and detergent removal was confirmed by mass spectrometry. We then performed negative-staining TEM on the protein-lipid dispersion and detected spherical or nearly spherical structures of diameters 40-370 nm (mean: 119 ± 63 nm, n = 108) corresponding to the proteoliposomes (Figure ). The RC reconstituted in GDGT membranes were next used for the spectroscopic studies. To ascertain that the RC is still intact and functional after reconstitution into archaeal lipid, we first recorded the UV-Vis spectra, which confirmed that the protein is still properly assembled, and that the chromophores are still in place (Figure ). Finally, we performed ultrafast transient absorption spectroscopy to see whether the RC is still able to perform primary electron transfer in this new environment. The results showed that RC can still undergo charge transfer, and the kinetics and mechanism are largely unchanged (Figure ).