Detection of Lipid-Bound Bacteriorhodopsin Trimer Complex Directly from Purple Membrane by Native Mass Spectrometry.

Native mass spectrometry (MS) was used to detect the membrane protein, bacteriorhodopsin (bR), in its 27 kDa monomeric form and trimeric assemblies directly from lipid-containing purple membranes (PMs) from the halophilic archaeon, Halobacterium salinarum. Trimer bR ion populations bound to lipid molecules were detected with n-octyl ß-d-glucopyranoside as the solubilizing detergent; the use of octyl tetraethylene glycol monooctyl ether or n-dodecyl-ß-d-maltopyranoside resulted in only detection of monomeric bR. The archaeal lipids phosphotidylglycerolphosphate methyl ester and 3-HSO3-Galp-ß1,6-Manp-α1,2-Glcp-α1,1-sn-2,3-diphytanylglycerol were the only lipids in the PMs found to bind to bR, consistent with previous high-resolution structural studies. Removal of the lipids from the sample resulted in the detection of only the bR monomer, highlighting the importance of specific lipids for stabilizing the bR trimer. To the best of our knowledge, this is the first report of the detection of the bR trimer with resolved lipid-bound species by MS.

Key determinants for signaling in the sensory rhodopsin II/transducer complex are different between Halobacterium salinarum and Natronomonas pharaonis.

The cell membrane of Halobacterium salinarum contains a retinal-binding photoreceptor, sensory rhodopsin II (HsSRII), coupled with its cognate transducer (HsHtrII), allowing repellent phototaxis behavior for shorter wavelength light. Previous studies on SRII from Natronomonas pharaonis (NpSRII) pointed out the importance of the hydrogen bonding interaction between Thr204NpSRII and Tyr174NpSRII in signal transfer from SRII to HtrII. Here, we investigated the effect on phototactic function by replacing residues in HsSRII corresponding to Thr204NpSRII and Tyr174NpSRII . Whereas replacement of either residue altered the photocycle kinetics, introduction of any mutations at Ser201HsSRII and Tyr171HsSRII did not eliminate negative phototaxis function. These observations imply the possibility of the presence of an unidentified molecular mechanism for photophobic signal transduction differing from NpSRII-NpHtrII.

Combined effect of the head groups and alkyl chains of archaea lipids when interacting with bacteriorhodopsin.

Bacteriorhodopsin (bR), a transmembrane protein with seven α-helices, is highly expressed in the purple membrane (PM) of archaea such as Halobacterium salinarum. It is well known that bR forms two-dimensional crystals with acidic lipids such as phosphatidylglycerol phosphate methyl ester (PGP-Me)-a major component of PM lipids bearing unique chemical structures-methyl-branched alkyl chains, ether linkages, and divalent anionic head groups with two phosphodiester groups. Therefore, we aimed to determine which functional groups of PGP-Me are essential for the boundary lipids of bR and how these functionalities interact with bR. To this end, we compared various well-known phospholipids (PLs) that carry one of the structural features of PGP-Me, and evaluated the affinity of PLs to bR using the centerband-only analysis of rotor-unsynchronized spin echo (COARSE) method in solid-state NMR measurements and thermal shift assays. The results clearly showed that the branched methyl groups of alkyl chains and double negative charges in the head groups are important for PL interactions with bR. We then examined the effect of phospholipids on the monomer-trimer exchange of bR using circular dichroism (CD) spectra. The results indicated that the divalent negative charge in a head group stabilizes the trimer structure, while the branched methyl chains significantly enhance the PLs' affinity for bR, thus dispersing bR trimers in the PM even at high concentrations. Finally, we investigated the effects of PL on the proton-pumping activity of bR based on the decay rate constant of the M intermediate of a bR photocycle. The findings showed that bR activities decreased to 20% in 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA), and in 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) bilayers as compared to that in PM. Meanwhile, 1,2-Diphytanoyl-sn-glycero-3-phosphate (DPhPA) bilayers bearing both negative charges and branched methyl groups preserved over 80% of the activity. These results strongly suggest that the head groups and alkyl chains of phospholipids are essential for boundary lipids and greatly influence the biological function of bR.

A conserved Trp residue in HwBR contributes to its unique tolerance toward acidic environments.

Bacteriorhodopsin (BR) is a light-driven outward proton pump found mainly in halophilic archaea. A BR from an archaeon Haloquadratum walsbyi (HwBR) was found to pump protons under more acidic conditions compared with most known BR proteins. The atomic structural study on HwBR unveiled that a pair of hydrogen bonds between the BC and FG loop in its periplasmic region may be a factor in such improved pumping capability. Here, we further investigated the retinal-binding pocket of HwBR and found that Trp94 contributes to the higher acid tolerance. Through single mutations in a BR from Halobacterium salinarum and HwBR, we examined the conserved tryptophan residues in the retinal-binding pocket. Among these residues of HwBR, mutagenesis at Trp94 facing the periplasmic region caused the most significant disruption to optical stability and proton-pumping capability under acidic conditions. The other tryptophan residues of HwBR exerted little impact on both maximum absorption wavelength and pH-dependent proton pumping. Our findings suggest that the residues from Trp94 to the hydrogen bonds at the BC loop confer both optical stability and functionality on the overall protein in low-pH environments.

Dynamic Coupling of Tyrosine 185 with the Bacteriorhodopsin Photocycle, as Revealed by Chemical Shifts, Assisted AF-QM/MM Calculations and Molecular Dynamic Simulations.

Aromatic residues are highly conserved in microbial photoreceptors and play crucial roles in the dynamic regulation of receptor functions. However, little is known about the dynamic mechanism of the functional role of those highly conserved aromatic residues during the receptor photocycle. Tyrosine 185 (Y185) is a highly conserved aromatic residue within the retinal binding pocket of bacteriorhodopsin (bR). In this study, we explored the molecular mechanism of the dynamic coupling of Y185 with the bR photocycle by automated fragmentation quantum mechanics/molecular mechanics (AF-QM/MM) calculations and molecular dynamic (MD) simulations based on chemical shifts obtained by 2D solid-state NMR correlation experiments. We observed that Y185 plays a significant role in regulating the retinal cis-trans thermal equilibrium, stabilizing the pentagonal H-bond network, participating in the orientation switch of Schiff Base (SB) nitrogen, and opening the F42 gate by interacting with the retinal and several key residues along the proton translocation channel. Our findings provide a detailed molecular mechanism of the dynamic couplings of Y185 and the bR photocycle from a structural perspective. The method used in this paper may be applied to the study of other microbial photoreceptors.

Stationary photocurrent generation from bacteriorhodopsin-loaded lipo-polymersomes in polyelectrolyte multilayer assembly on polyethersulfone membrane.

Vesicles constructed of either synthetic polymers alone (polymersomes) or a combination of polymers and lipids (lipo-polymersomes) demonstrate excellent long-term stability and ability to integrate membrane proteins. Applications using lipo-polymersomes with integrated membrane proteins require suitable supports to maintain protein functionality. Using lipo-polymersomes loaded with the light-driven proton pump bacteriorhodopsin (BR), we demonstrate here how the photocurrent is influenced by a chosen support. In our study, we deposited BR-loaded lipo-polymersomes in a cross-linked polyelectrolyte multilayer assembly either directly physisorbed on gold electrode microchips or cross-linked on an intermediary polyethersulfone (PES) membrane covalently grafted using a hydrogel cushion. In both cases, electrochemical impedance spectroscopic characterization demonstrated successful polyelectrolyte assembly with BR-loaded lipo-polymersomes. Light-induced proton pumping by BR-loaded lipo-polymersomes in the different support constructs was characterized by amperometric recording of the generated photocurrent. Application of the hydrogel/PES membrane support together with the polyelectrolyte assembly decreased the transient current response upon light activation of BR, while enhancing the generated stationary current to over 700 nA/cm2. On the other hand, the current response from BR-loaded lipo-polymersomes in a polyelectrolyte assembly without the hydrogel/PES membrane support was primarily a transient peak combined with a low-nanoampere-level stationary photocurrent. Hence, the obtained results demonstrated that by using a hydrogel/PES support it was feasible to monitor continuously light-induced proton flux in biomimetic applications of lipo-polymersomes. Graphical abstract.

How Does a Microbial Rhodopsin RxR Realize Its Exceptionally High Thermostability with the Proton-Pumping Function Being Retained?

We often encounter a case where two proteins, whose amino-acid sequences are similar, are quite different with regard to the thermostability. As a striking example, we consider the two seven-transmembrane proteins: recently discovered Rubrobacter xylanophilus rhodopsin (RxR) and long-known bacteriorhodopsin from Halobacterium salinarum (HsBR). They commonly function as a light-driven proton pump across the membrane. Though their sequence similarity and identity are ∼71 and ∼45%, respectively, RxR is much more thermostable than HsBR. In this study, we solve the three-dimensional structure of RxR using X-ray crystallography and find that the backbone structures of RxR and HsBR are surprisingly similar to each other: The root-mean-square deviation for the two structures calculated using the backbone Cα atoms of the seven helices is only 0.86 Å, which makes the large stability difference more puzzling. We calculate the thermostability measure and its energetic and entropic components for RxR and HsBR using our recently developed statistical-mechanical theory. The same type of calculation is independently performed for the portions playing essential roles in the proton-pumping function, helices 3 and 7, and their structural properties are related to the probable roles of water molecules in the proton-transporting mechanism. We succeed in elucidating how RxR realizes its exceptionally high stability with the original function being retained. This study provides an important first step toward the establishment of a method correlating microscopic, geometric characteristics of a protein with its thermodynamic properties and enhancing the thermostability through amino-acid mutations without vitiating the original function.

Archaeal Glycolipid S-TGA-1 Is Crucial for Trimer Formation and Photocycle Activity of Bacteriorhodopsin.

Although it has been demonstrated that membrane proteins (MPs) require lipids to ensure their structural and functional integrity, details on how lipid-MP interactions regulate MPs are still unclear. Recently, we developed a concise method for quantitatively evaluating lipid-MP interactions and applied it to bacteriorhodopsin (bR), a halobacterial MP that forms trimers and acts as a light-driven proton pump. Consequently, we found that the halobacterial glycolipid, S-TGA-1, has the highest affinity for bR, among other lipids. In this study, we examined the effects of S-TGA-1 on bR via visible circular dichroism spectroscopy, flash photolysis, and proton influx measurement. The results showed that S-TGA-1 efficiently promotes trimer formation, photocycle, and proton pumping in bR. Our data also suggested that the bR photocycle is restored as a consequence of the trimerization induced by the lipid. This study demonstrates clearly that lipids specifically interacting with MPs can have significant impacts on MP structure and/or function. The methodology adopted in our studies can be applied to other MPs and will help elucidate the physiological functions of lipids in terms of lipid-MP interactions, thus accelerating "lipid chemical biology" studies.

First-Principles Characterization of the Elusive I Fluorescent State and the Structural Evolution of Retinal Protonated Schiff Base in Bacteriorhodopsin.

The conversion of light energy into work is essential to life on earth. Bacteriorhodopsin (bR), a light-activated proton pump in Archae, has served for many years as a model system for the study of this process in photoactive proteins. Upon absorption of a photon, its chromophore, the retinal protonated Schiff base (RPSB), isomerizes from its native all-trans form to a 13-cis form and pumps a proton out of the cell in a process that is coupled to eventual ATP synthesis. Despite numerous time-resolved spectroscopic studies over the years, the details of the photodynamics of bR on the excited state, particularly the characterization of the I fluorescent state, the time-resolved reaction mechanism, and the role of the counterion cluster of RPSB, remain uncertain. Here, we use ab initio multiple spawning (AIMS) with spin-restricted ensemble Kohn-Sham (REKS) theory to simulate the nonadiabatic dynamics of the ultrafast photoreaction in bR. The excited state dynamics can be partitioned into three distinct phases: (1) relaxation away from the Franck-Condon region dominated by changes in retinal bond length alternation, (2) dwell time on the excited state in the I fluorescent state featuring an untwisted, bond length inverted RPSB, and (3) rapid torsional evolution to the conical intersection after overcoming a small excited state barrier. We fully characterize the I fluorescent state and the excited state barrier that hinders direct evolution to the conical intersection following photoexcitation. We also find that photoisomerization is accompanied by weakening of the interaction between RPSB and its counterion cluster. However, in contradiction with a recent time-resolved X-ray experiment, hydrogen bond cleavage is not necessary to reproduce the observed photoisomerization dynamics.

Settling the Long-Standing Debate on the Proton Storage Site of the Prototype Light-Driven Proton Pump Bacteriorhodopsin.

Despite decades of research, the location and molecular identity of the proton release group together with the subsequent proton release pathway remain controversial even for the simplest light-driven proton pump, bacteriorhodopsin, according to the most recent experiments and simulations. Yet despite this nagging lack of knowledge for the generic case, even more complex pumps are currently under investigation. The proton release group disclosed by our large-scale simulations satisfies available experimental results, especially the broad Zundel continuum absorption subject to a striking anisotropy observed only recently. Moreover, our simulations delineate the seamless pathway by which the excess proton (being stored in an ultrastrong centered H-bond involving two glutamates) is finally translocated into the extracellular medium.

Remarkably enhanced photoelectrical efficiency of bacteriorhodopsin in quantum dot - Purple membrane complexes under two-photon excitation.

The photosensitive protein bacteriorhodopsin (bR) has been shown to be a promising material for optoelectronic applications, but it cannot effectively absorb and utilize light energy in the near-infrared (NIR) region of the optical spectrum. Semiconductor quantum dots (QDs) have two-photon absorption cross-sections two orders of magnitude larger than those of bR and can effectively transfer the up-converted energy of two NIR photons to bR via the Förster resonance energy transfer (FRET). In this study, we have engineered a photoelectrochemical cell based on a hybrid material consisting of QDs and bR-containing purple membranes (PMs) of Halobacterium salinarum and demonstrated that this cell can generate an electrical signal under the two-photon laser excitation. We have shown that the efficiency of light conversion by the PM-QD hybrid material under two-photon excitation is up to 4.3 times higher than the efficiency of conversion by PMs alone. The QD integration into the bR-containing PMs significantly improves the bR capacity for utilizing light upon two-photon laser excitation, thus paving the way to the engineering of biologically inspired hybrid NIR nonlinear optoelectronic elements. The nonlinear nature of two-photon excitation may provide considerable advantages, such as a sharp sensitivity threshold and the possibility of precise three-dimensional location of excitation in holography and optical computing.

Bacteriorhodopsin: Structural Insights Revealed Using X-Ray Lasers and Synchrotron Radiation.

Directional transport of protons across an energy transducing membrane-proton pumping-is ubiquitous in biology. Bacteriorhodopsin (bR) is a light-driven proton pump that is activated by a buried all-trans retinal chromophore being photoisomerized to a 13-cis conformation. The mechanism by which photoisomerization initiates directional proton transport against a proton concentration gradient has been studied by a myriad of biochemical, biophysical, and structural techniques. X-ray free electron lasers (XFELs) have created new opportunities to probe the structural dynamics of bR at room temperature on timescales from femtoseconds to milliseconds using time-resolved serial femtosecond crystallography (TR-SFX). Wereview these recent developments and highlight where XFEL studies reveal new details concerning the structural mechanism of retinal photoisomerization and proton pumping. We also discuss the extent to which these insights were anticipated by earlier intermediate trapping studies using synchrotron radiation. TR-SFX will open up the field for dynamical studies of other proteins that are not naturally light-sensitive.

A concise method for quantitative analysis of interactions between lipids and membrane proteins.

Although interactions between lipids and membrane proteins (MPs) have been considered crucially important for understanding the functions of lipids, lack of useful and convincing experimental methods has hampered the analysis of the interactions. Here, we developed a surface plasmon resonance (SPR)-based concise method for quantitative analysis of lipid-MP interactions, coating the sensor chip surface with self-assembled monolayer (SAM) with C6-chain. To develop this method, we used bacteriorhodopsin (bR) as an MP, and examined its interaction with various types of lipids. The merits of using C6-SAM-modified sensor chip are as follows: (1) alkyl-chains of SAM confer a better immobilization of MPs because of the efficient preconcentration due to hydrophobic contacts; (2) SAM provides immobilized MPs with a partial membranous environment, which is important for the stabilization of MPs; and (3) a thinner C6-SAM layer (1 nm) compared with MP size forces the MP to bulge outward from the SAM surface, allowing extraneously injected lipids to be accessible to the hydrophobic transmembrane regions. Actually, the amount of bR immobilized on C6-SAM is 10 times higher than that on a hydrophilic CM5 sensor chip, and AFM observations confirmed that bR molecules are exposed on the SAM surface. Of the lipids tested, S-TGA-1, a halobacterium-derived glycolipid, had the highest specificity to bR with a nanomolar dissociation constant. This is consistent with the reported co-crystal structure that indicates the formation of several intermolecular hydrogen bonds. Therefore, we not only reproduced the specific lipid-bR recognition, but also succeeded in its quantitative evaluation, demonstrating the validity and utility of this method.

Photochemistry of Bacteriorhodopsin with Various Oligomeric Statuses in Controlled Membrane Mimicking Environments: A Spectroscopic Study from Femtoseconds to Milliseconds.

Preparing transmembrane protein in controllable lipid bilayers is essential for unravelling the coupling of the environments and its dynamic functions. Monomerized bacteriorhodopsin (mbR) embedded in covalently circularized nanodiscs was prepared with dimyristoylphosphatidylglycerol (DMPG) lipid and circular membrane scaffold proteins of two different sizes, cE3D1 and cΔ H5, respectively. The retinal photoisomerization kinetics and thermodynamic photocycle were examined by femtosecond and nanosecond transient absorption, respectively, covering the time scale from femtoseconds to hundreds of milliseconds. The kinetics of the retinal isomerization and proton migration from the protonated Schiff base to Asp-85 were not significantly different for monomeric bR solubilized in Triton X-100 or embedded in circularized nanodiscs. This can be ascribed to the local tertiary structures at the retinal pocket vicinity being similar among monomeric bR in various membrane mimicking environments. However, the aforementioned processes are intrinsically different for trimeric bR in purple membrane (PM) and delipidated PM. The reprotonation of the deprotonated Schiff base from Asp-96 in association with the decay of intermediate M, which involved wide-ranged structural alteration, manifested a difference in terms of the oligomeric statuses, as well as a slight dependence on the size of the nanodisc. In summary, bR oligomeric statuses, rather than the environmental factors, such as membrane mimicking systems and nanodisc size, play a significant role in bR photocycle associated with short-range processes, such as the retinal isomerization and deprotonation of protonated Schiff base at the retinal pocket. On the other hand, the environmental factors, such as the types of membrane mimicking systems and the size of nanodiscs, affect those dynamic processes involving wider structural alterations during the photocycle.

Sulfated archaeol glycolipids: Comparison with other immunological adjuvants in mice.

Archaeosomes are liposomes traditionally comprised of total polar lipids (TPL) or semi-synthetic glycerolipids of ether-linked isoprenoid phytanyl cores with varied glyco- and amino-head groups. As adjuvants, they induce robust, long-lasting humoral and cell-mediated immune responses and enhance protection in murine models of infectious disease and cancer. Traditional total polar lipid (TPL) archaeosome formulations are relatively complex and first generation semi-synthetic archaeosomes involve many synthetic steps to arrive at the final desired glycolipid composition. We have developed a novel archaeosome formulation comprising a sulfated disaccharide group covalently linked to the free sn-1 hydroxyl backbone of an archaeal core lipid (sulfated S-lactosylarchaeol, SLA) that can be more readily synthesized yet retains strong immunostimulatory activity for induction of cell-mediated immunity following systemic immunization. Herein, we have evaluated the immunostimulatory effects of SLA archaeosomes when used as adjuvant with ovalbumin (OVA) and hepatitis B surface antigen (HBsAg) and compared this to various other adjuvants including TLR3/4/9 agonists, oil-in-water and water-in-oil emulsions and aluminum hydroxide. Overall, we found that semi-synthetic sulfated glycolipid archaeosomes induce strong Ag-specific IgG titers and CD8 T cells to both antigens. In addition, they induce the expression of a number of cytokines/chemokines including IL-6, G-CSF, KC & MIP-2. SLA archaeosome formulations demonstrated strong adjuvant activity, superior to many of the other tested adjuvants.

Fast and high-resolution mapping of elastic properties of biomolecules and polymers with bimodal AFM.

Fast, high-resolution mapping of heterogeneous interfaces with a wide elastic modulus range is a major goal of atomic force microscopy (AFM). This goal becomes more challenging when the nanomechanical mapping involves biomolecules in their native environment. Over the years, several AFM-based methods have been developed to address this goal. However, none of these methods combine sub-nanometer spatial resolution, quantitative accuracy, fast data acquisition speed, wide elastic modulus range and operation in physiological solutions. Here, we present detailed procedures for generating high-resolution maps of the elastic properties of biomolecules and polymers using bimodal AFM. This requires the simultaneous excitation of the first two eigenmodes of the cantilever. An amplitude modulation (AM) feedback acting on the first mode controls the tip-sample distance, and a frequency modulation (FM) feedback acts on the second mode. The method is fast because the elastic modulus, deformation and topography images are obtained simultaneously. The method is efficient because only a single data point per pixel is needed to generate the aforementioned images. The main stages of the bimodal imaging are sample preparation, calibration of the instrument, tuning of the microscope and generation of the nanomechanical maps. In addition, with knowledge of the deformation, bimodal AFM enables reconstruction of the true topography of the surface. It takes ~9 h to complete the whole procedure.

Resonance Raman Investigation of the Chromophore Structure of Heliorhodopsins.

Heliorhodopsins (HeRs) are a new category of retinal-bound proteins recently discovered through functional metagenomics analysis that exhibit obvious differences from type-1 microbial rhodopsins. We conducted the first detailed structural characterization of the retinal chromophore in HeRs using resonance Raman spectroscopy. The observed spectra clearly show that the Schiff base of the chromophore is protonated and forms a strong hydrogen bond to a species other than a water molecule, highly likely a counterion residue. The vibrational mode of the Schiff base of HeRs exhibits similarities with that of photosensory microbial rhodopsins, that is consistent with the previous proposal that HeRs function as photosensors. We also revealed unusual spectral features of the in-plane chain vibrations of the chromophore, suggesting an unprecedented geometry of the Schiff base caused by a difference in the retinal pocket structure of HeRs. These data demonstrate structural characteristics of the photoreceptive site in this novel type of rhodopsin family.

Highly Efficient Transfer of 7TM Membrane Protein from Native Membrane to Covalently Circularized Nanodisc.

Incorporating membrane proteins into membrane mimicking systems is an essential process for biophysical studies and structure determination. Monodisperse lipid nanodiscs have been found to be a suitable tool, as they provide a near-native lipid bilayer environment. Recently, a covalently circularized nanodisc (cND) assembled with a membrane scaffold protein (MSP) in circular form, instead of conventional linear form, has emerged. Covalently circularized nanodiscs have been shown to have improved stability, however the optimal strategies for the incorporation of membrane proteins, as well as the physicochemical properties of the membrane protein embedded in the cND, have not been studied. Bacteriorhodopsin (bR) is a seven-transmembrane helix (7TM) membrane protein, and it forms a two dimensional crystal consisting of trimeric bR on the purple membrane of halophilic archea. Here it is reported that the bR trimer in its active form can be directly incorporated into a cND from its native purple membrane. Furthermore, the assembly conditions of the native purple membrane nanodisc (PMND) were optimized to achieve homogeneity and high yield using a high sodium chloride concentration. Additionally, the native PMND was demonstrated to have the ability to assemble over a range of different pHs, suggesting flexibility in the preparation conditions. The native PMND was then found to not only preserve the trimeric structure of bR and most of the native lipids in the PM, but also maintained the photocycle function of bR. This suggests a promising potential for assembling a cND with a 7TM membrane protein, extracted directly from its native membrane environment, while preserving the protein conformation and lipid composition.

X-ray structure analysis of bacteriorhodopsin at 1.3 Å resolution.

Bacteriorhodopsin (bR) of Halobacterium salinarum is a membrane protein that acts as a light-driven proton pump. bR and its homologues have recently been utilized in optogenetics and other applications. Although the structures of those have been reported so far, the resolutions are not sufficient for elucidation of the intrinsic structural features critical to the color tuning and ion pumping properties. Here we report the accurate crystallographic analysis of bR in the ground state. The influence of X-rays was suppressed by collecting the data under a low irradiation dose at 15 K. Consequently, individual atoms could be separately observed in the electron density map at better than 1.3 Å resolution. Residues from Thr5 to Ala233 were continuously constructed in the model. The twist of the retinal polyene was determined to be different from those in the previous models. Two conformations were observed for the proton release region. We discuss the meaning of these fine structural features.

Using a portable Raman spectrometer to detect carotenoids of halophilic prokaryotes in synthetic inclusions in NaCl, KCl, and sulfates.

Cell suspensions of the haloarchaea Halorubrum sodomense and Halobacterium salinarum and the extremely halophilic bacterium Salinibacter ruber (Bacteroidetes) in saturated solutions of chlorides and sulfates (NaCl, KCl, MgSO4·7H2O, K2SO4, and (NH4)Al(SO4)2·12H2O) were left to evaporate to produce micrometric inclusions in laboratory-grown crystals. Raman spectra of these pinkish inclusions were obtained using a handheld Raman spectrometer with green excitation (532 nm). This portable instrument does not include any microscopic tool. Acceptable Raman spectra of carotenoids were obtained in the range of 200-4000 cm-1. This detection achievement was related to the mode of illumination and collection of scattered light as well as due to resonance Raman enhancement of carotenoid signals under green excitation. The position of diagnostic Raman carotenoid bands corresponds well to those specific carotenoids produced by a given halophile. To our best knowledge, this is the first study of carotenoids included in the laboratory in crystalline chlorides and sulfates, using a miniature portable Raman spectrometer. Graphical abstract ᅟ.