Bacteriorhodopsin Crystals Research Articles

Demonstration of electron diffraction from membrane protein crystals grown in a lipidic mesophase after lamella preparation by focused ion beam milling at cryogenic temperatures.

Electron crystallography of sub-micrometre-sized 3D protein crystals has emerged recently as a valuable field of structural biology. In meso crystallization methods, utilizing lipidic mesophases, particularly lipidic cubic phases (LCPs), can produce high-quality 3D crystals of membrane proteins (MPs). A major step towards realizing 3D electron crystallography of MP crystals, grown in meso, is to demonstrate electron diffraction from such crystals. The first task is to remove the viscous and sticky lipidic matrix that surrounds the crystals without damaging the crystals. Additionally, the crystals have to be thin enough to let electrons traverse them without significant multiple scattering. In the present work, the concept that focused ion beam milling at cryogenic temperatures (cryo-FIB milling) can be used to remove excess host lipidic mesophase matrix is experimentally verified, and then the crystals are thinned to a thickness suitable for electron diffraction. In this study, bacteriorhodopsin (BR) crystals grown in a lipidic cubic mesophase of monoolein were used as a model system. LCP from a part of a hexagon-shaped plate-like BR crystal (∼10 µm in thickness and ∼70 µm in the longest dimension), which was flash-frozen in liquid nitro-gen, was milled away with a gallium FIB under cryogenic conditions, and a part of the crystal itself was thinned into a ∼210 nm-thick lamella with the ion beam. The frozen sample was then transferred into an electron cryo-microscope, and a nanovolume of ∼1400 × 1400 × 210 nm of the BR lamella was exposed to 200 kV electrons at a fluence of ∼0.06 e Å-2. The resulting electron diffraction peaks were detected beyond 2.7 Å resolution (with an average peak height to background ratio of >2) by a CMOS-based Ceta 16M camera. The results demonstrate that cryo-FIB milling produces high-quality lamellae from crystals grown in lipidic mesophases and pave the way for 3D electron crystallography on crystals grown or embedded in highly viscous media.

An Electronic and Optically Controlled Bifunctional Transistor Based on a Bio-Nano Hybrid Complex.

We report an electronically and optically controlled bioelectronic field-effect transistor (FET) based on the hybrid film of photoactive bacteriorhodopsin and electronically conducting single-walled carbon nanotubes (SWNTs). Two-dimensional (2D) crystals of bacteriorhodopsin form the photoactive center of the bio–nano complex, whereas one-dimensional (1D) pure SWNTs provide the required electronic support. The redshift in the Raman spectra indicates the electronic doping with an estimated charge density of 3 × 106 cm–2. The hybrid structure shows a conductivity of 19 μS/m and semiconducting characteristics due to preferential binding with selective diameters of semiconducting SWNTs. The bioelectronic transistor fabricated using direct laser lithography shows both optical and electronic gating with a significant on/off switch ratio of 8.5 and a photoconductivity of 13.15 μS/m. An n-type FET shows complementary p-type characteristics under light due to optically controlled, electronic doping by the “proton-pumping” bacteriorhodopsin. The fabricated bioelectronic transistor exhibits both electronically and optically well-controlled bifunctionality based on the functionalized hybrid electronic material.

The Power of Freeze-Fracture Electron Microscopy in Membrane Exploration

Freeze-fracture transmission electron microscopy (ff-TEM) as a cryo-fixation, replica technique enables us to monitor highly dynamic nano-scale events in micro-scale biological as well as artificial membrane assemblies. Furthermore, freeze-fracturing allows a unique look at the interior of cell membranes because the fracture plane follows the area of weakest forces and splitting membrane bilayers into two opposite, complementary sheets. Therefore it is still the only way to obtain detailed structural information about lateral inhomogeneities in cell as well as model membranes in a probe-free mode. Applying this unique technique we are able to study formation and transformation of lipid-, protein-, toxin-, as well as drug micro domains. Examples will be given for lipid domains in liposomal bilayer and their transformation by temperature, incorporation of amphiphilic drugs, and proteins. Furthermore, domain initiation by toxin and phage protein will be shown. Lipid-induced modulation of 2-D crystals of bacteriorhodopsin in liposomal bilayer demonstrates an extreme example for domain formation of membrane-spanning proteins. Additionally to domain explorations, we were able to study unique membranes made of bola-type Archaebacteria lipids and to identify transitions between their horseshoe- and membrane-spanning arrangements. Furthermore applying this unique TEM technique, we investigated transformations to non-bilayer structures such as hexagonal and cubic phases.

Low-Z polymer sample supports for fixed-target serial femtosecond X-ray crystallography

X-ray free-electron lasers (XFELs) offer a new avenue to the structural probing of complex materials, including biomolecules. Delivery of precious sample to the XFEL beam is a key consideration, as the sample of interest must be serially replaced after each destructive pulse. The fixed-target approach to sample delivery involves depositing samples on a thin-film support and subsequent serial introduction via a translating stage. Some classes of biological materials, including two-dimensional protein crystals, must be introduced on fixed-target supports, as they require a flat surface to prevent sample wrinkling. A series of wafer and transmission electron microscopy (TEM)-style grid supports constructed of low-Z plastic have been custom-designed and produced. Aluminium TEM grid holders were engineered, capable of delivering up to 20 different conventional or plastic TEM grids using fixed-target stages available at the Linac Coherent Light Source (LCLS). As proof-of-principle, X-ray diffraction has been demonstrated from two-dimensional crystals of bacteriorhodopsin and three-dimensional crystals of anthrax toxin protective antigen mounted on these supports at the LCLS. The benefits and limitations of these low-Z fixed-target supports are discussed; it is the authors' belief that they represent a viable and efficient alternative to previously reported fixed-target supports for conducting diffraction studies with XFELs.

Crystal structure of the O intermediate of the Leu93→Ala mutant of bacteriorhodopsin

The lifetime of the O intermediate of bacteriorhodopsin (BR) is extended by a factor of ∼250 in the Leu93-to-Ala mutant (BR_L93A). To clarify the structural changes occurring in the last stage of the proton pumping cycle of BR, we crystallized BR_L93A into a hexagonal P622 crystal. Diffraction data from the unphotolyzed state showed that the deletion of three carbon atoms from Leu93 is compensated by the insertion of four water molecules in the cytoplasmic vicinity of retinal. This insertion of water is suggested to be responsible for the blue-shifted λ(max) (540 nm) of the mutant. A long-lived substate of O with a red-shifted λ(max) (~565 nm) was trapped when the crystal of BR_L93A was flash-cooled after illumination with green light. This substate (O(slow)) bears considerable similarity to the M intermediate of native BR; that is, it commonly shows deformation of helix C and the FG loop, downward orientation of the side chain of Arg82, and disruption of the Glu194/Glu204 pair. In O(slow), however, the main chain of Lys216 is less distorted and retinal takes on the 13-cis/15-syn configuration. Another significant difference is seen in the pH dependence of the structure of the proton release group, the pK(a) value of which is suggested to be much lower in O(slow) than in M.

Controlled in meso phase crystallization--a method for the structural investigation of membrane proteins.

We investigated in meso crystallization of membrane proteins to develop a fast screening technology which combines features of the well established classical vapor diffusion experiment with the batch meso phase crystallization, but without premixing of protein and monoolein. It inherits the advantages of both methods, namely (i) the stabilization of membrane proteins in the meso phase, (ii) the control of hydration level and additive concentration by vapor diffusion. The new technology (iii) significantly simplifies in meso crystallization experiments and allows the use of standard liquid handling robots suitable for 96 well formats. CIMP crystallization furthermore allows (iv) direct monitoring of phase transformation and crystallization events. Bacteriorhodopsin (BR) crystals of high quality and diffraction up to 1.3 Å resolution have been obtained in this approach. CIMP and the developed consumables and protocols have been successfully applied to obtain crystals of sensory rhodopsin II (SRII) from Halobacterium salinarum for the first time.

Electrostatic effects influence the formation of two-dimensional crystals of bacteriorhodopsin reconstituted into dimyristoylphosphatidylcholine membranes

We examined how electrostatic shielding affects the formation of two-dimensional (2D) crystals of bacteriorhodopsin (bR) in reconstituted dimyristoylphosphatidylcholine (DMPC) membranes by varying the sodium chloride (NaCl) concentration. The 2D crystalline array of bR formed in the gel phase of DMPC membranes was characterized by a symmetric bipolar pattern in visible circular dichroic spectra collected around 560 nm. The amplitude of the bipolar pattern was systematically enhanced by increasing the NaCl concentration. A strong correlation between the amplitude of the bipolar pattern and the Debye constant of small ions indicated that a weakening of electrostatic repulsion by the shielding effect of small ions enhances the order of 2D bR crystals in the gel phase of DMPC membranes. Considering the 3D distribution of charged residues, we propose a model of interaction balance in reconstituted bR membranes in which effective attraction between bR molecules occurs as a result of the phase separation of the DMPC membrane in the gel phase overcoming electrostatic repulsion between bR molecules.

Detection of Membrane Protein Two-Dimensional Crystals in Living Cells

It is notoriously difficult to grow membrane protein crystals and solve membrane protein structures. Improved detection and screening of membrane protein crystals are needed. We have shown here that second-order nonlinear optical imaging of chiral crystals based on second harmonic generation can provide sensitive and selective detection of two-dimensional protein crystalline arrays with sufficiently low background to enable crystal detection within the membranes of live cells. The method was validated using bacteriorhodopsin crystals generated in live Halobacterium halobium bacteria and confirmed by electron microscopy from the isolated crystals. Additional studies of alphavirus glycoproteins indicated the presence of localized crystalline domains associated with virus budding from mammalian cells. These results suggest that in vivo crystallization may provide a means for expediting membrane protein structure determination for proteins exhibiting propensities for two-dimensional crystal formation.

Isoprenoid-chained lipid β-XylOC 16+4—A novel molecule for in meso membrane protein crystallization

Here we report a successful use of a recently developed isoprenoid-chained lipid family for in meso crystallization of membrane proteins. The isoprenoid-chained lipid 1-O-(3,7,11,15-tetramethylhexadecyl)-β– d-xyloside (β-XylOC 16+4) used as a host lipid for in meso crystallization provided high quality bacteriorhodopsin (bR) crystals (P6 3 space group) diffracting to high resolution and characterized by low twinning ratio. β-XylOC 16+4 has an isoprenoid chain with methyl branches at each 4th position and a xylose group in the water-soluble part. These peculiarities make the lipid clearly distinguishable in the bR crystalline lattice and provides a unique opportunity to study the role of the host lipid in the in meso crystallization. We conclude that β-XylOC 16+4 may have a general application for in meso crystallization for a wide range of membrane proteins. The cubic phase of β-XylOC 16+4 is present over a wide range of temperatures and is stable at low temperature (down to about 8 °C). This opens up the possibility of using temperature as a tool for the optimization of in meso crystallization with additional advantages for the crystallization of membrane proteins at lower temperatures where the proteins of interest may be more stable.

Zernike Phase Contrast Cryo-Electron Microscopy and Tomography for Structure Determination at Nanometer and Subnanometer Resolutions

Zernike phase contrast cryo-electron microscopy (ZPC-cryoEM) is an emerging technique that is capable of producing higher image contrast than conventional cryoEM. By combining this technique with advanced image processing methods, we achieved subnanometer resolution for two biological specimens: 2D bacteriorhodopsin crystal and epsilon15 bacteriophage. For an asymmetric reconstruction of epsilon15 bacteriophage, ZPC-cryoEM can reduce the required amount of data by a factor of approximately 3, compared with conventional cryoEM. The reconstruction was carried out to 13 A resolution without the need to correct the contrast transfer function. New structural features at the portal vertex of the epsilon15 bacteriophage are revealed in this reconstruction. Using ZPC cryo-electron tomography (ZPC-cryoET), a similar level of data reduction and higher resolution structures of epsilon15 bacteriophage can be obtained relative to conventional cryoET. These results show quantitatively the benefits of ZPC-cryoEM and ZPC-cryoET for structural determinations of macromolecular machines at nanometer and subnanometer resolutions.

Adenosine Receptor Modeling: What Does the A2A Crystal Structure Tell Us?

For a long time, there have been no experimentally determined structural data for any adenosine receptor (AR) and the only approach available for making structure/function correlations about these proteins has been homology modeling. While the early attempts to model these receptors followed the crystallization of bacteriorhodopsin, the cryo-microscopy studies of bovine and frog rhodopsin, and the modeling of a Calpha-template for the TM helices in the rhodopsin family of GPCRs, the crystallization of bovine rhodopsin by Palczewski was of extreme importance as it first provided the crystal structure of an eukaryotic GPCR to be used as template for more realistic homology models. Since then, rhodopsin-based modeling became the routine approach to develop AR structural models that proved to be useful for interpretation of site-directed mutagenesis data and for molecular docking studies. The recently reported crystal structures of the adrenergic beta1 and beta2 receptors only partially confirmed the structural features showed by bovine rhodopsin, raising a question about which template would have been better for further modeling of ARs. Such question remained actually not-answered, due to the publication in late 2008 of the crystal structure of human adenosine A(2A) receptor (AA(2A)R). Since its publication, this structure has been used for ligands docking analysis and has provided a high similarity template for homology modeling of the other AR subtypes. Still, the AA(2A)R crystal structure allows to verify the hypotheses that were made on the basis of the previously reported homology modeling and molecular docking.

Overcoming merohedral twinning in crystals of bacteriorhodopsin grown in lipidic mesophase

Twinning is one of the most common crystal-growth defects in protein crystallography. There are neither efficient rational approaches for the growth of nontwinned protein crystals nor are there examples of systematic studies of the dependence of the twinning-ratio distribution on crystallization conditions. The description of the twinning phenomenon has been covered even less for membrane-protein crystals and is non-existent for crystals grown using lipidic phases (in meso). In the present work, possibilities for overcoming merohedral twinning are investigated for crystals of the membrane protein bacteriorhodopsin (bR) grown in meso. It is shown that traditional crystallization additives are not effective in the case of the in meso crystallization of bR. The twinning ratio was determined for 310 crystals grown under different crystallization conditions. A correlation of the twinning ratio with the growth rate of the crystals was observed. Slow growth indicated that crystals had a noticeable chance of avoiding twinning. Model calculations were performed in order to rationalize this observation. The calculations confirmed the experimental observation that most crystals consist of two twin domains and showed that under this condition small changes in the probability of twin-domain formation lead to dramatic changes in the number of nontwinned crystals, which explains why slow crystal growth results in a considerable number of nontwinned crystals.

A Plug-Based Microfluidic System for Dispensing Lipidic Cubic Phase (LCP) Material Validated by Crystallizing Membrane Proteins in Lipidic Mesophases.

This paper presents a plug-based microfluidic system to dispense nanoliter-volume plugs of Lipidic Cubic Phase (LCP) material and subsequently merge the LCP plugs with aqueous plugs. This system was validated by crystallizing membrane proteins in lipidic mesophases, including LCP. This system allows for accurate dispensing of LCP material in nanoliter volumes, prevents inadvertent phase transitions that may occur due to dehydration by enclosing LCP in plugs, and is compatible with the traditional method of forming LCP material using a membrane protein sample, as shown by the successful crystallization of bacteriorhodopsin from Halobacterium salinarum. Conditions for the formation of LCP plugs were characterized and presented in a phase diagram. This system was also implemented using two different methods of introducing the membrane protein: 1) the traditional method of generating the LCP material using a membrane protein sample and 2) Post LCP-formation Incorporation (PLI), which involves making LCP material without protein, adding the membrane protein sample externally to the LCP material, and allowing the protein to diffuse into the LCP material or into other lipidic mesophases that may result from phase transitions. Crystals of bacterial photosynthetic reaction centers from Rhodobacter sphaeroides and Blastochloris viridis were obtained using PLI. The plug-based, LCP-assisted microfluidic system, combined with the PLI method for introducing membrane protein into LCP, should be useful for minimizing consumption of samples and broadening the screening of parameter space in membrane protein crystallization.

Crystal Structures of Different Substates of Bacteriorhodopsin's M Intermediate at Various pH Levels

The hexagonal P622 crystal of bacteriorhodopsin, which is made up of stacked membranes, is stable provided that the precipitant concentration in the soaking solution is higher than a critical value (i.e., 1.5 M ammonium sulfate). Diffraction data showed that the crystal lattice shrank linearly with increasing precipitant concentration, due primarily to narrowing of intermembrane spaces. Although the crystal shrinkage did not affect the rate of formation of the photoreaction M intermediate, its lifetime increased exponentially with the precipitant concentration. It was suggested that the energetic barrier of the M-to-N transition becomes higher when the motional freedom of the EF loop is reduced by crystal lattice force. As a result of this property, the M state accumulated predominantly when the crystal that was soaked at a high precipitant concentration was illuminated at room temperature. Structural data obtained at various pH levels showed that the overall structure of M is not strongly dependent on pH, except that Glu194 and Glu204 in the proton release complex are more separated at pH 7 than at pH 4.4. This result suggests that light-induced disruption of the paired structure of Glu194 and Glu204 is incomplete when external pH is lower than the pK(a) value of the proton release group in the M state.

Domain/Raft Exploration in Lipid Mono- & Bilayer by Freeze-fracture Electron Microscopy on Nano-Resolution Scale

Lateral chemical and physical inhomogeneities of biological membranes such as domains/rafts seem to play an important role in signal transduction, membrane traffic, and diseases.Freeze-fracture electron microscopy (ff-em) as a cryofixation TEM technique is a powerful to explore such small and highly dynamic domains in a probe-free mode. Since the resolution of this technique is 2 nm we are able to study lipid-, protein-, toxin-, as well as drug domains on a nano-resolution scale. Since replica, resistant to beam damage, can be produced from large, micro-meter size objects, ff-em allows us to study nano-scale events in micro-scale biological as well as artificial assemblies. The fact that the fracture plane follows the area of weakest forces, allows insides into the hydrophobic center of lipid bilayer [1-3] as well as into the lipid/gas interface of lipid monolayer stabilizing gas bubbles [4].Examples will be given for domains in liposomal bilayer made of drugs [5], proteins, and toxin. Lipid-induced modulation of 2-D crystals of bacteriorhodopsin in liposomal bilayer will be shown as an extreme example for domain formation of intrinsic proteins [6-8]. Additionally, liquid ordered (Lo) domains will be shown recently detected in lipid monolayer, stabilizing hydrophobic gas bubbles.[1] B. Sternberg, Liposome Technology, CRC Press I (1992) 363.[2] B. Sternberg, Handbook Nonmedical Applications of Liposomes CRC Press (1996) 271.[3] B. Sternberg, Medical Applications of Liposomes, Elsevier (1998) 395.[4] C. Brancewicz et al. J. Disp. Sci. & Techn. 27:5 (2006) 761.[5] K. Merz and B. Sternberg J. Drug Targ. 2 (1994) 411.[6] B. Sternberg et al. Biochim. Biophys. Acta 980 (1989) 117.[7] B. Sternberg et al. Biochim. Biophys. Acta 1108 (1992) 21.[8] B. Sternberg et al. J. Struc. Biol. 110 (1993) 196.

Comparative analysis of the quality of membrane protein bacteriorhodopsin crystals during crystallization in octylglucoside and octylthioglucoside

Crystallization of bacteriorhodopsin (bR) in the lipidic cubic phase using n-octyl-β-D-glucoside (OG) and its more stable and inexpensive analogue n-octyl-β-D-thioglucoside (OTG) was comparatively analyzed [1]. It was shown that bacteriorhodopsin is efficiently crystallized in OTG in the same detergent concentration range as in OG. However, x-ray diffraction analysis shows that bR crystals in OG are characterized by a better resolution (1.35 A) than bR crystals in OTG (1.45 A).

Interactions of lipids with aquaporin-0 and other membrane proteins

The structure of aquaporin-0 (AQP0) has recently been determined by electron crystallography of two-dimensional (2D) crystals and by X-ray crystallography of three-dimensional (3D) crystals. The electron crystallographic structure revealed nine lipids per AQP0 monomer, which form an almost complete bilayer. The lipids adopt a wide variety of conformations and tightly fill the space between adjacent AQP0 tetramers. The conformations of the lipid acyl chains appear to be determined not only by the protein surface but also by the acyl chains of adjacent lipid molecules. In the X-ray structure, the hydrophobic region of the protein is surrounded by a detergent micelle, with two ordered detergent molecules per AQP0 monomer. Despite the different environments, the electron crystallographic and X-ray structures of AQP0 are virtually identical, but they differ in the temperature factors of the atoms that either contact the lipids in the 2D crystals or are exposed to detergents in the 3D crystals. The temperature factors are higher in the X-ray structure, suggesting that the detergent-exposed AQP0 residues are less ordered than the corresponding ones contacting lipids in the 2D crystals. An examination of ordered detergent molecules in crystal structures of other aquaporins and of lipid molecules in 2D and 3D crystals of bacteriorhodopsin suggests that the increased conformational variability of detergent-exposed residues compared to lipid-contacting residues is a general feature.

Determination of astigmatism in TEM images

We have developed a new two-step algorithm to determine the astigmatism of images from transmission electron microscopes (TEMs). Instead of computing the radial average of the power spectrum, we divide the power spectrum of a TEM image 1 to m (typically 32) sectors. We use a technique based on perturbation analysis of the contrast transfer function (CTF) to assimilate sector averages of the power spectrum of an image, which are incoherent in the presence of astigmatism, to a coherent radial average corresponding to a nominal defocus value. This is based on the fact that small defocus change from a nominal value can be considered to be equivalent to a perturbation on the spatial frequency spectra. Thus, instead of measuring the angular defocus variations, we optimise the frequency change required to obtain a coherent radial average. Numerically, this is achieved by minimizing σ 2/ σ 1 of a matrix formed from the sector averages, where σ i denotes the ith singular value of the matrix. After the minimisation procedure, the second singular value should be very small compared with the first singular value, indicating that the matrix is nearly rank unity. In the second step, the nominal defocus can be obtained from the coherent radial average using any good defocus estimation program, which assumes zero astigmatism. The defocus value at a sector can be obtained from this nominal defocus value and one of the parameters from the unconstrained optima. Our algorithm is tested on astigmatic images of carbon film, 2D crystals of bacteriorhodopsin and cryo-images of HIV cores.

Time-Resolved Microspectroscopy on a Single Crystal of Bacteriorhodopsin Reveals Lattice-Induced Differences in the Photocycle Kinetics

The determination of the intermediate state structures of the bacteriorhodopsin photocycle has lead to an unprecedented level of understanding of the catalytic process exerted by a membrane protein. However, the crystallographic structures of the intermediate states are only relevant if the working cycle is not impaired by the crystal lattice. Therefore, we applied visible and Fourier transform infrared spectroscopy (FTIR) microspectroscopy with microsecond time resolution to compare the photoreaction of a single bacteriorhodopsin crystal to that of bacteriorhodopsin residing in the native purple membrane. The analysis of the FTIR difference spectra of the resolved intermediate states reveals great similarity in structural changes taking place in the crystal and in PM. However, the kinetics of the photocycle are significantly altered in the three-dimensional crystal as compared to PM. Strikingly, the L state decay is accelerated in the crystal, whereas the M decay is delayed. The physical origin of this deviation and the implications for trapping of intermediate states are discussed. As a methodological advance, time-resolved step-scan FTIR spectroscopy on a single protein crystal is demonstrated for the first time which may be used in the future to gauge the functionality of other crystallized proteins with the molecular resolution of vibrational spectroscopy.

High-resolution powder diffraction study of purple membrane with a large Guinier-type camera

X-ray diffraction patterns from a film of oriented purple membranes, which comprise two-dimensional crystals of bacteriorhodopsin (BR) trimers, were recorded with a 1 m-pathlength Guinier-type camera at SPring-8 BL40B2. A well focused X-ray beam and a camera with a high angular resolution of 0.024 degrees enabled a powder diffraction profile with very sharp and well separated peaks to be obtained up to a resolution of 2.3 A. Using integrated diffraction intensities up to a Bragg spacing of 4.2 A, a cluster of bulky amino acid residues and the head group of the BR chromophore are apparent in the electron density map projected along the membrane normal. Thus, a combination of synchrotron X-rays and large Guinier camera can be used for analyzing the conformational changes of BR in the intact state. In addition, the method might be extended to the structural analysis of film materials composed of two-dimensional arrays of nanoparticles.