In Situ Study of the Function of Bacterioruberin in the Dual‐Chromophore Photoreceptor Archaerhodopsin‐4

Abstract

While certain archaeal ion pumps have been shown to contain two chromophores, retinal and the carotenoid bacterioruberin, the functions of bacterioruberin have not been well explored. To address this research gap, recombinant archaerhodopsin-4 (aR4), either with retinal only or with both retinal and bacterioruberin chromophores, was successfully expressed together with endogenous lipids in H. salinarum L33 and MPK409 respectively. In situ solid-state NMR, supported by molecular spectroscopy and functional assays, revealed for the first time that the retinal thermal equilibrium in the dark-adapted state is modulated by bacterioruberin binding through a cluster of aromatic residues on helix E. Bacterioruberin not only stabilizes the protein trimeric structure but also affects the photocycle kinetics and the ATP formation rate. These new insights may be generalized to other receptors and proteins in which metastable thermal equilibria and functions are perturbed by ligand binding.

Oriented Nanoarchitectonics of Bacteriorhodopsin for Enhancing ATP Generation in a FoF1‐ATPase‐Based Assembly System

“…To confirm that the adsorption procedure was successful, the ξ potential of each layer was measured (Figure 2c). The UV/Vis absorption spectrum of BR within the microcapsules is similar to that of BR in solution, [11] which indicates BR was successfully loaded into the microcapsules (Figure S1d).…”

Solid-state NMR structure determination of a membrane protein in E. coli cellular inner membrane

“…Therefore, determining the MP structures in their native cellular membrane environment is critical to precisely reveal MP structures in physiological membranes and to validate structures determined in artificial membrane mimetics. ssNMR can characterize biomolecule structures in native cellular membranes (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26), so-called cellular ssNMR (13). ssNMR has been used to study MPs in Escherichia coli cellular membranes (11,12,(14)(15)(16)(17)(18)20) and purple membranes (19,27) and the organization of cellular context (13).…”

“…ssNMR can characterize biomolecule structures in native cellular membranes (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26), so-called cellular ssNMR (13). ssNMR has been used to study MPs in Escherichia coli cellular membranes (11,12,(14)(15)(16)(17)(18)20) and purple membranes (19,27) and the organization of cellular context (13). These studies have used either signal enhancements by sample preparation (13,16,28) and NMR spectral approaches, such as dynamic nuclear polarization (DNP) (14,(29)(30)(31) and 1 H detection (32,33), or background signal suppressions by using deleted strains (13) and antibiotics (34).…”

De novo resonance assignment of the transmembrane domain of LR11/SorLA in E. coli membranes

“…Both isolated membrane vesicles (in situ) and intact cells (in-cell) can be used effectively with MAS ssNMR. With targeted sample preparation aimed at reducing the signals of background lipids and proteins [6,7] and 1 H-detection and dynamic nuclear polarization (DNP) techniques to improve spectral sensitivity [8][9][10][11][12][13][14][15][16][17], in situ and in cell ssNMR have provided unique insights into the effects of complex biological membranes on the structure and dynamics of membrane proteins and their complexes [18][19][20][21][22][23][24]. For example, ssNMR studies of M2 [25], DAGK [26], PagL [27], Mistic [16], CsmA [28], ASR (Anabaena sensory rhodopsin) [7], and Aquaporin Z [29] in biological membranes validated structures that were previously determined using model membrane systems.…”