Nanostructured 7-9-residue cyclic and unstructured lipopeptide-based facial detergents have been engineered to stabilize the model integral membrane protein, bacteriorhodopsin. Formation of a cylindrical-type micelle assembly induced by facial amphipathic lipopeptides resembles a biological membrane more effectively than conventional micelles. The hydrophobic face of this cylindrical-type micelle provides extended stability to the membrane protein and the hydrophilic surface interacts with an aqueous environment. In our present study, we have demonstrated experimentally and computationally that lipopeptide-based facial detergents having an unstructured or β-turn conformation can stabilize membrane proteins. However, constrained peptide detergents can provide enhanced stability to bacteriorhodopsin. In this study, we have computationally examined the structural stability of bacteriorhodopsin in the presence of helical, beta-strand, and cyclic unstructured peptide detergents, and conventional detergent-like peptides. Our study demonstrates that optimal membranomimetics (detergents) for stabilizing a specific membrane protein can be screened based on the following criteria: (i) hydrodynamic radii of the self-assembled peptide detergents, (ii) stability assay of detergent-encased membrane proteins, (iii) percentage covered area of detergent-encased membrane proteins obtained computationally and (iv) protein-detergent interaction energy.

The photoinduced all-trans to 13-cis isomerization of the retinal Schiff base represents the ultrafast first step in the reaction cycle of bacteriorhodopsin (BR). Extensive experimental and theoretical work has addressed excited-state dynamics and isomerization via a conical intersection with the ground state. In conflicting molecular pictures, the excited state potential energy surface has been modeled as a pure S[Formula: see text] state that intersects with the ground state, or in a 3-state picture involving the S[Formula: see text] and S[Formula: see text] states. Here, the photoexcited system passes two crossing regions to return to the ground state. The electric dipole moment of the Schiff base in the S[Formula: see text] and S[Formula: see text] state differs strongly and, thus, its measurement allows for assessing the character of the excited-state potential. We apply the method of ultrafast terahertz (THz) Stark spectroscopy to measure electric dipole changes of wild-type BR and a BR D85T mutant upon electronic excitation. A fully reversible transient broadening and spectral shift of electronic absorption is induced by a picosecond THz field of several megavolts/cm and mapped by a 120-fs optical probe pulse. For both BR variants, we derive a moderate electric dipole change of 5 [Formula: see text] 1 Debye, which is markedly smaller than predicted for a neat S[Formula: see text]-character of the excited state. In contrast, S[Formula: see text]-admixture and temporal averaging of excited-state dynamics over the probe pulse duration gives a dipole change in line with experiment. Our results support a picture of electronic and nuclear dynamics governed by the interaction of S[Formula: see text] and S[Formula: see text] states in a 3-state model.

We report herein the synthesis of three detergents bearing a perfluorinated cyclohexyl group connected through a short, hydrogenated spacer (i.e., propyl, butyl, or pentyl) to a β-maltoside polar head that are, respectively, called FCymal-3, FCymal-4, and FCymal-5. Increasing the length of the spacer decreased the critical micellar concentration (CMC), as demonstrated by surface tension (SFT) and isothermal titration calorimetry (ITC), from 5 mM for FCymal-3 to 0.7 mM for FCymal-5. The morphology of the micelles was studied by dynamic light scattering (DLS), analytical ultracentrifugation (AUC), and small-angle X-ray scattering (SAXS), indicating heterogeneous rod-like shapes. While micelles of FCymal-3 and -4 have similar hydrodynamic diameters of ∼10 nm, those of FCymal-5 were twice as large. We also investigated the ability of the detergents to solubilize lipid membranes made of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC). Molecular modeling indicated that the FCymal detergents generate disorder in lipid bilayers, with FCymal-3 being inserted more deeply into bilayers than FCymal-4 and -5. This was experimentally confirmed using POPC vesicles that were completely solubilized within 2 h with FCymal-3, whereas FCymal-5 required >8 h. A similar trend was noticed for the direct extraction of membrane proteins from E. coli membranes, with FCymal-3 being more potent than FCymal-5. An opposite trend was observed in terms of stabilization of the two model membrane proteins bacteriorhodopsin (bR) and SpNOX. In all three FCymal detergents, bR was stable for at least 2 months with no signs of aggregation. However, while the structural integrity of bR was fully preserved in FCymal-4 and -5, minor bleaching was observed in FCymal-3. Similarly, SpNOX exhibited the least activity in FCymal-3 and the highest activity in FCymal-5. By combining solubilizing and stabilizing potency, FCymal detergents push forward our expectations of the usefulness of fluorinated detergents for handling and investigating membrane proteins.

Liquid crystal (LC) biosensors have received significant attention for their potential applications for point-of-care devices due to their sensitivity, low cost, and easy read-out. They have been employed to detect a wide range of important biological molecules. However, detecting the function of membrane proteins has been extremely challenging due to the difficulty of integrating membrane proteins, lipid membranes, and LCs into one system. In this study, we addressed this challenge by monitoring the proton-pumping function of bacteriorhodopsin (bR) using a pH-sensitive LC thin film biosensor. To achieve this, we deposited purple membranes (PMs) containing a 2D crystal form of bRs onto an LC-aqueous interface. Under light, the PM patches changed the local pH at the LC-aqueous interface, causing a color change in the LC thin film that is observable through a polarizing microscope with crossed polarizers. These findings open up new opportunities to study the biofunctions of membrane proteins and their induced local environmental changes in a solution using LC biosensors.

The article correlates between symmetry breaking and phase transition. An analogy, extending from physics to biology, is known to exist between these two topics. Bacteriorhodopsin (bR) as a paradigm of membrane proteins has been used as a case study in the present work. The bR, as the sole protein embedded in what is called a purple membrane (PM), has attracted widespread interest in bionanotechnological applications. The lipids of PM have a crucial role in maintaining the crystal lattice of bR inside PM. For this reason, the present work has been concerned with elucidating the thermal phase transition properties of the PM lipids in orthogonal directions. The results indicated that the axial symmetry of bR exhibits considerable changes occurring at the thermal phase transition of lipids. These changes are brought by an anomaly observed in the time course of orthogonal electric responses during the application of thermal fields on PM. The observed anomaly may bear on symmetry breaking in bR occurring at the phase transition of lipids based on such analogy found between symmetry breaking and phase transition. Lipid-protein interactions may underlie the broken axial symmetry of bR at such lipid thermal transition of PM. Accordingly, thermally perturbed axial symmetry of bR may be of biological relevance relying on the essence of the crystal lattice of bR. Most importantly, a question has to be raised in the present study: Can bR, as a helical protein with broken axial symmetry, affect the symmetry breaking of helical light? This may be of potential technical applications based on a recent discovery that bR breaks the symmetry of helical light.

Membrane protein folding is distinct from folding of soluble proteins. Conformational acquisition in major membrane protein subclasses can be delineated into insertion and folding processes. An exception to the \"two stage\" folding, later developed to \"three stage\" folding, is observed within the last two helices in bacteriorhodopsin (BR), a system that serves as a model membrane protein. We employ a reductionist approach to understand interplay of molecular factors underlying the apparent defiance. Leveraging available solution NMR structures, we construct, sample in silico, and analyze partially (PIn) and fully inserted (FIn) BR membrane states. The membrane lateral C-terminal helix (CH) in PIn is markedly prone to transient structural distortions over microsecond timescales; a disorder prone region (DPR) is thereby identified. While clear transmembrane propensities are not acquired, the distortions induce alterations in local membrane curvature and area per lipid. Importantly, energetic decompositions reveal that overall, the N-terminal helix (NH) is thermodynamically more stable in the PIn. Higher overall stability of the FIn arises from favorable interactions between the NH and the CH. Our results establish lack of spontaneous transition of the PIn to the FIn, and attributes their partitioning to barriers that exceed those accessible with thermal fluctuations. This work paves the way for further detailed studies aimed at determining the thermo-kinetic roles of the initial five helices, or complementary external factors, in complete helical folding and insertion in BR. We comment that complementing such efforts with the growing field of machine learning assisted energy landscape searches may offer unprecedented insights.

Helical membrane proteins generally have a hydrophobic nature, with apolar side chains comprising the majority of the transmembrane (TM) helices. However, whenever polar side chains are present in the TM domain, they often exert a crucial role in structural interactions with other polar residues, such as TM helix associations and oligomerization. Moreover, polar residues in the TM region also often participate in protein functions, such as the Schiff base bonding between Lys residues and retinal in rhodopsin-like membrane proteins. Although many studies have focused on these functional polar residues, our understanding of stand-alone polar residues that are energetically unfavored in TM helixes is limited. Here, we adopted bacteriorhodopsin (bR) as a model system and systematically mutated 17 of its apolar Leu or Phe residues to polar Asn. Stability measurements of the resulting mutants revealed that all of these polar substitutions reduced bR stability to various extents, and the extent of destabilization of each mutant bR is also correlated to different structural factors, such as the relative accessible surface area and membrane depth of the mutation site. Structural analyses of these Asn residues revealed that they form sidechain-to-backbone hydrogen bonds that alleviate the unfavorable energetics in hydrophobic and apolar surroundings. Our results indicate that membrane proteins are able to accommodate certain stand-alone polar residues in the TM region without disrupting overall structures.

Discovered over 50 years ago, bacteriorhodopsin is the first recognized and most widely studied microbial retinal protein. Serving as a light-activated proton pump, it represents the archetypal ion-pumping system. Here we compare the photochemical dynamics of bacteriorhodopsin light and dark-adapted forms with that of the first metastable photocycle intermediate known as \"K\". We observe that following thermal double isomerization of retinal in the dark from bio-active all-trans 15-anti to 13-cis, 15-syn, photochemistry proceeds even faster than the ~0.5 ps decay of the former, exhibiting ballistic wave packet curve crossing to the ground state. In contrast, photoexcitation of K containing a 13-cis, 15-anti chromophore leads to markedly multi-exponential excited state decay including much slower stages. QM/MM calculations, aimed to interpret these results, highlight the crucial role of protonation, showing that the classic quadrupole counterion model poorly reproduces spectral data and dynamics. Single protonation of ASP212 rectifies discrepancies and predicts triple ground state structural heterogeneity aligning with experimental observations. These findings prompt a reevaluation of counter ion protonation in bacteriorhodopsin and contribute to the broader understanding of its photochemical dynamics.

Boundary lipids surrounding membrane proteins play an essential role in protein function and structure. These protein-lipid interactions are mainly divided into electrostatic interactions between the polar amino acids of proteins and polar heads of phospholipids, and hydrophobic interactions between protein transmembrane sites and phospholipid acyl chains. Our previous report (Kawatake et al., Biochim. Biophys. Acta 1858 [2016] 2106-2115) covered a method for selectively analyzing boundary lipid interactions and showed differences in membrane protein-peripheral lipid interactions due to differences in their head group. Interactions in the hydrophobic acyl chains of phospholipids are relatively consistent among proteins, but the details of these interactions have not been elucidated. In this study, we reconstituted bacteriorhodopsin as a model protein into phospholipid membranes labeled with 2H and 13C for solid-state NMR measurement to investigate the depth-dependent effect of the head group structure on the lipid bilayer. The results showed that the position of the phospholipid near the carbonyl carbon was affected by the head group in terms of selectivity for protein surfaces, whereas in the deep interior of the bilayer near the leaflet interface, there was little difference between the head groups, indicating that the dependence of their interactions on the head group was much reduced.

Controlling the pH at the microliter scale can be useful for applications in research, medicine, and industry, and therefore represents a valuable application for synthetic biology and microfluidics. The presented vesicular system translates light of different colors into specific pH changes in the surrounding solution. It works with the two light-driven proton pumps bacteriorhodopsin and blue light-absorbing proteorhodopsin Med12, that are oriented in opposite directions in the lipid membrane. A computer-controlled measuring device implements a feedback loop for automatic adjustment and maintenance of a selected pH value. A pH range spanning more than two units can be established, providing fine temporal and pH resolution. As an application example, a pH-sensitive enzyme reaction is presented where the light color controls the reaction progress. In summary, light color-controlled pH-adjustment using engineered proteoliposomes opens new possibilities to control processes at the microliter scale in different contexts, such as in synthetic biology applications.