Crystallization of bacteriorhodopsin from bicelle formulations at room temperature

We showed previously that high-quality crystals of bacteriorhodopsin (bR) from Halobacterium salinarum can be obtained from bicelle-forming DMPC/CHAPSO mixtures at 37 degrees C. As many membrane proteins are not sufficiently stable for crystallization at this high temperature, we tested whether the bicelle method could be applied at a lower temperature. Here we show that bR can be crystallized at room temperature using two different bicelle-forming compositions: DMPC/CHAPSO and DTPC/CHAPSO. The DTPC/CHAPSO crystals grown at room temperature are essentially identical to the previous, twinned crystals: space group P21 with unit cell dimensions of a = 44.7 A, b = 108.7 A, c = 55.8 A, beta = 113.6 degrees . The room-temperature DMPC/CHAPSO crystals are untwinned, however, and belong to space group C222(1) with the following unit cell dimensions: a = 44.7 A, b = 102.5 A, c = 128.2 A. The bR protein packs into almost identical layers in the two crystal forms, but the layers stack differently. The new untwinned crystal form yielded clear density for a previously unresolved CHAPSO molecule inserted between protein subunits within the layers. The ability to grow crystals at room temperature significantly expands the applicability of bicelle crystallization.     

Crystallization of bacteriorhodopsin solubilized by a tripod amphiphile

Bacteriorhodopsin (bR) is solubilized efficiently as a monomer by a novel surfactant, a tripod amphiphile (TPA), which permits the formation of purple hexagonal bR crystals under several conditions. The crystals, although small, diffract to 2.5 A resolution using synchrotron radiation. TPA may be useful for the solubilization, purification, and crystallization of other membrane proteins.     

A new approach for structure analysis of two-dimensional membrane protein crystals using X-ray powder diffraction data

The application of powder diffraction methods to problems in structural biology is generally regarded as intractable because of the large number of unresolved, overlapping X‐ray reflections. Here, we use information about unit cell lattice parameters, space group transformations, and chemical composition as a priori information in a bootstrap process that resolves the ambiguities associated with overlapping reflections. The measured ratios of reflections that can be resolved experimentally are used to refine the position, the shape, and the orientation of low‐resolution molecular structures within the unit cell, in leading to the resolution of the overlapping reflections. The molecular model is then made progressively more sophisticated as additional diffraction information is included in the analysis. We apply our method to the recovery of the structure of the bacteriorhodopsin molecule (bR) to a resolution of 7 Å using experimental data obtained from two‐dimensional purple membrane crystals. The approach can be used to determine the structure factors directly or to provide reliable low‐resolution phase information that can be refined further by the conventional methods of protein crystallography.     

Protein, lipid and water organization in bacteriorhodopsin crystals: a molecular view of the purple membrane at 1.9 A resolution.

BACKGROUND: Bacteriorhodopsin (bR) from Halobacterium salinarum is a proton pump that converts the energy of light into a proton gradient that drives ATP synthesis. The protein comprises seven transmembrane helices and in vivo is organized into purple patches, in which bR and lipids form a crystalline two-dimensional array. Upon absorption of a photon, retinal, which is covalently bound to Lys216 via a Schiff base, is isomerized to a 13-cis,15-anti configuration. This initiates a sequence of events - the photocycle - during which a proton is transferred from the Schiff base to Asp85, followed by proton release into the extracellular medium and reprotonation from the cytoplasmic side. RESULTS: The structure of bR in the ground state was solved to 1.9 A resolution from non-twinned crystals grown in a lipidic cubic phase. The structure reveals eight well-ordered water molecules in the extracellular half of the putative proton translocation pathway. The water molecules form a continuous hydrogen-bond network from the Schiff-base nitrogen (Lys216) to Glu194 and Glu204 and includes residues Asp85, Asp212 and Arg82. This network is involved both in proton translocation occurring during the photocycle, as well as in stabilizing the structure of the ground state. Nine lipid phytanyl moieties could be modeled into the electron-density maps. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of single crystals demonstrated the presence of four different charged lipid species. CONCLUSIONS: The structure of protein, lipid and water molecules in the crystals represents the functional entity of bR in the purple membrane of the bacteria at atomic resolution. Proton translocation from the Schiff base to the extracellular medium is mediated by a hydrogen-bond network that involves charged residues and water molecules.     

Crystallization and preliminary X-ray diffraction analysis of a plant ribonuclease from the seeds of the bitter gourd Momordica charantia.

Single crystals of ribonuclease Mc, a new class of plant ribonuclease from the seeds of the bitter gourd, were obtained from solutions of polyethylene glycol 8000 by the hanging-drop vapour diffusion method. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 67.28 A, b = 75.21 A, c = 38.54 A. The assumption of one monomer per asymmetric unit gives rise to a Vm value of 2.29 A3/Da. The crystals diffract beyond 2.0 A resolution and are suitable for high resolution X-ray structure analysis.     

Use of a fusion protein to obtain crystals suitable for X-ray analysis: crystallization of a GST-fused protein containing the DNA-binding domain of DNA replication-related element-binding factor, DREF.

Crystals of glutathione-S-transferase (GST)-fused protein containing the DNA-binding domain of DNA replication-related element-binding factor, DREF, were obtained under crystallization conditions similar to those for GST. Preliminary X-ray crystallographic analysis revealed that crystals of the GST-fused protein belong to space group P6(1)22 or P6(5)22 with unit cell dimensions a = b = 140.4 A, c = 93.5 A and gamma = 120 degrees, having one molecule in the crystallographic asymmetric unit. The crystals diffract to 2.5 A resolution. The cell dimensions are related to those of GST crystals thus far reported. Crystallization of the DNA-binding domain that was cleaved from the fused protein by thrombin was also carried out using several methods under numerous conditions, but efforts to produce well-ordered large crystals were unsuccessful. A possible application of GST-fusion proteins for small target proteins or domains to obtain crystals suitable for X-ray structure determination is proposed.     

Crystallization and the crystal properties of the oxygen-evolving photosystem II from Synechococcus vulcanus

A photosystem II (PSII) complex highly active in oxygen evolution was purified and crystallized from a thermophilic cyanobacterium, Synechococcus vulcanus. The PSII complex in the crystals contained the D1/D2 reaction center subunits, CP47 and CP43 (two chlorophyll-binding core antenna proteins of photosystem II), cytochrome b-559 alpha- and beta-subunits, several low molecular weight subunits, and three extrinsic proteins, that is, 33 and 12 kDa proteins and cytochrome c-550. The PSII complex also retained a high rate of oxygen evolution. The apparent molecular mass of the PSII in the crystals was determined to be 580 kDa by gel filtration chromatography, indicating that the PSII crystallized is a dimer. The crystals diffracted to a maximum resolution of 3.5 A at a cryogenic temperature using X-rays from a synchrotron radiation source, SPring-8. The crystals belonged to an orthorhombic system, and the space group was P2(1)2(1)2(1) with unit cell dimensions of a = 129.7 A, b = 226.5 A, and c = 307.8 A. Each asymmetric unit contained one PSII dimer, which gave rise to a specific volume (V(M)) of 3.6 A(3)/Da based on the calculated molecular mass of 310 kDa for a PSII monomer and an estimated solvent content of 66%. Multiple data sets of native crystals have been collected and processed to 4.0 A, indicating that our crystals are suitable for structure analysis at this resolution.     

Membrane-protein stability in a phospholipid-based crystallization medium

Protein stability is a crucial factor to consider when attempting to crystallize integral membrane proteins. Cubic phase, or in meso, lipid-bilayer crystallization media are thought to provide native-like environments that should facilitate membrane protein crystallization by helping to stabilize the native protein conformation for the duration of the crystallization process. While excellent crystals of bacteriorhodopsin (bR) and other Halobacterial rhodopsins have been obtained in lipid-bilayer gels formed with monoglycerides, success remains elusive in the general application of such media to other membrane proteins. Additionally, we have noted that some mutants of bR are highly unstable in gels formed with monoolein. Phosphatidylethanolamines (PE) and derivatives of PE represent another class of lipids that can form connected-bilayer gels. When wildtype bR and a labile bR mutant were reconstituted into this phospholipid gel, spectroscopy showed that the protein is both more stable and has improved conformational homogeneity as compared to gels formed using monoolein. In addition, we demonstrate that well-diffracting crystals of bR can be grown from a PE-based crystallization medium. Since most proteins lack a stability-indicating chromophore and other structure-based analytical techniques are poorly compatible with the lipid gel, we developed a generally-applicable spectroscopic technique based on the intrinsic fluorescence of tryptophan residues. This fluorescence assay makes possible the rapid evaluation of lipid gels as media for the crystallization of membrane proteins.     

2-D structure of the Neurospora crassa plasma membrane ATPase as determined by electron cryomicroscopy.

Large, well-ordered 2-D crystals of the dodecylmaltoside complex of the Neurospora crassa plasma membrane H(+)-ATPase grow rapidly on the surface of a polyethylene glycol-containing mixture similar to that originally developed for growing 3-D crystals of this integral membrane transport protein. Negative stain electron microscopy of the crystals shows that many are single layers. Cryoelectron microscopy of unstained specimens indicates that the crystals have a p6 layer group with unit cell dimensions of a = b = 167 A. Image processing of selected electron micrographs has yielded a projection map at 10.3 A resolution. The repeating unit of the ATPase crystals comprises six 100 kDa ATPase monomers arranged in a symmetrical ring. The individual monomers in projection are shaped like a boot. These results provide the first indications of the molecular structure of the H(+)-ATPase molecule. They also establish the feasibility of precipitant-induced surface growth as a rapid, simple alternative to conventional methods for obtaining 2-D crystals of the integral membrane proteins useful for structure analysis.     

An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin

Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR) is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example. Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression. This complementary protein approach allowed us to increase bR expression by two orders of magnitude through the introduction of two silent mutations into bR coding DNA. For the first time the high quality crystals of bR expressed in E. Coli were obtained using the produced protein. The crystals obtained with in meso nanovolume crystallization diffracted to 1.67 Å.     

Crystal structure of deltarhodopsin‐3 from Haloterrigena thermotolerans

Deltarhodopsin, a new member of the microbial rhodopsin family, functions as a light‐driven proton pump. Here, we report the three‐dimensional structure of deltarhodopsin (dR3) from Haloterrigena thermotolerans at 2.7 Å resolution. A crystal belonging to space group R32 (a, b = 111.71 Å, c = 198.25 Å) was obtained by the membrane fusion method. In this crystal, dR3 forms a trimeric structure as observed for bacteriorhodopsin (bR). Structural comparison of dR with bR showed that the inner part (the proton release and uptake pathways) is highly conserved. Meanwhile, residues in the protein–protein contact region are largely altered so that the diameter of the trimeric structure at the cytoplasmic side is noticeably larger in dR3. Unlike bR, dR3 possesses a helical segment at the C‐terminal region that fills the space between the AB and EF loops. A significant difference is also seen in the FG loop, which is one residue longer in dR3. Another peculiar property of dR3 is a highly crowded distribution of positively charged residues on the cytoplasmic surface, which may be relevant to a specific interaction with some cytoplasmic component.Proteins 2013; © 2013 Wiley Periodicals, Inc.     

Use of detergents in two-dimensional crystallization of membrane proteins

Structure determination at high resolution is actually a difficult challenge for membrane proteins and the number of membrane proteins that have been crystallized is still small and far behind that of soluble proteins. Because of their amphiphilic character, membrane proteins need to be isolated, purified and crystallized in detergent solutions. This makes it difficult to grow the well-ordered three-dimensional crystals that are required for high resolution structure analysis by X-ray crystallography. In this difficult context, growing crystals confined to two dimensions (2D crystals) and their structural analysis by electron crystallography has opened a new way to solve the structure of membrane proteins. However, 2D crystallization is one of the major bottlenecks in the structural studies of membrane proteins. Advances in our understanding of the interaction between proteins, lipids and detergents as well as development and improvement of new strategies will facilitate the success rate of 2D crystallization. This review deals with the various available strategies for obtaining 2D crystals from detergent-solubilized intrinsic membrane proteins. It gives an overview of the methods that have been applied and gives details and suggestions of the physical processes leading to the formation of the ordered arrays which may be of help for getting more proteins crystallized in a form suitable for high resolution structural analysis by electron crystallography.     

X-ray diffraction from membrane protein nanocrystals

Membrane proteins constitute >30% of the proteins in an average cell, and yet the number of currently known structures of unique membrane proteins is <300. To develop new concepts for membrane protein structure determination, we have explored the serial nanocrystallography method, in which fully hydrated protein nanocrystals are delivered to an x-ray beam within a liquid jet at room temperature. As a model system, we have collected x-ray powder diffraction data from the integral membrane protein Photosystem I, which consists of 36 subunits and 381 cofactors. Data were collected from crystals ranging in size from 100 nm to 2 μm. The results demonstrate that there are membrane protein crystals that contain <100 unit cells (200 total molecules) and that 3D crystals of membrane proteins, which contain <200 molecules, may be suitable for structural investigation. Serial nanocrystallography overcomes the problem of x-ray damage, which is currently one of the major limitations for x-ray structure determination of small crystals. By combining serial nanocrystallography with x-ray free-electron laser sources in the future, it may be possible to produce molecular-resolution electron-density maps using membrane protein crystals that contain only a few hundred or thousand unit cells.     

Crystallization and preliminary X-ray characterization of maltoporin from Escherichia coli

Crystals of maltoporin (the bacteriophage lambda receptor of Escherichia coli) that diffract X-rays to 3 A resolution can be grown reproducibly. Maltoporin is an integral membrane protein, which forms a channel in the E. coli outer membrane that specifically facilitates the diffusion of maltose and maltodextrins. The crystals have a rhombic prismatic habit and belong to the orthorhombic space group C222(1) with unit cell dimensions a = 130 A, b = 213 A and c = 216 A. X-ray structure determination is underway.     

Crystallization and structure determination of goat lactoferrin at 4.0 A resolution: a new form of packing in lactoferrins with a high solvent content in crystals

Lactoferrin was purified from fresh samples of goat colostrums, saturated with Fe3+ and CO3(2-) ions and crystallized by microdialysis method. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with a=104.6 A, b=153.8 A, c=155.1 A and Z=4. The quality of crystals was poor, thus the intensity data were restricted to 4.0 A resolution only. The structure was determined by molecular replacement method using diferric buffalo lactoferrin as a model. The solution clearly indicated the presence of one molecule in the asymmetric unit, which corresponds to a Vm value of 7.1 A3/Da. The structure was refined with stringent constraints to an R-factor of 0.246 using all the reflections 15,870 to 4.0 A resolution. The overall structure of goat lactoferrin is essentially similar to those of buffalo and bovine lactoferrins. However, the iron-binding environment in goat lactoferrin is somewhat different, in which 2 CO3(2-). ions have low occupancies. The solvent content of approximately 84% was very high in the present case which explains the fragility of the crystals of goat lactoferrin. In a way, it is very surprising that the crystals grow at all, although crystals with solvent as high as 89% have been reported.     

Two-dimensional crystallization of Escherichia coli-expressed bacteriorhodopsin and its D96N variant: high resolution structural studies in projection.

Highly ordered two-dimensional (2-D) crystals of Escherichia coli-expressed bacteriorhodopsin analog (e-bR) and its D96N variant (e-D96N) reconstituted in Halobacterium halobium lipids have been obtained by starting with the opsin protein purified in the denaturing detergent sodium dodecyl sulfate. These crystals embedded in glucose show electron diffraction in projection to better than 3.0 A at room temperature. This is the first instance that expressed bR or a variant has been crystallized in 2-D arrays showing such high order. The crystal lattice is homologous to that in wild-type bR (w-bR) in purple membranes (PM) and permit high resolution analyses of the structure of the functionally impaired D96N variant. The e-bR crystal is isomorphous to that in PM with an overall averaged fractional change of 12.7% (26-3.6-A resolution) in the projection structure factors. The projection difference Fourier map e-bR-PM at 3.6-A resolution indicates small conformational changes equivalent to movement of approximately < 7 C-atoms distributed within and in the neighborhood of the protein envelope. This result shows that relative to w-bR there are no global structural rearrangements in e-bR at this 3.6 A resolution level. The e-D96N crystal is isomorphous to the e-bR crystal with a smaller (9.2%) overall averaged fractional change in the structure factors. The significant structural differences between e-D96N and e-bR are concentrated at high resolution (5-3.6 A); however, these changes are small as quantified from the 3.6 A resolution e-D96N-e-bR Fourier difference map. The difference map showed no statistically significant peaks or valleys within 5 A in projection from the site of D96 substitution on helix C. Elsewhere within the protein envelope the integrated measure of peaks or valleys was < approximately 3 C-atom equivalents. Thus, our results show that for the isosteric substitution of Asp96 by Asn, the molecular conformation of bR in its ground state is essentially unaltered. Therefore, the known effect of D96N on the slowed M412 decay is not due to ground-state structural perturbations.     

Purification and crystallization of benzoylformate decarboxylase.

A new large-scale purification method for benzoylformate decarboxylase from Pseudomonas putida has allowed us to undertake an X-ray crystallographic study of the enzyme. The previously observed instability of the enzyme was overcome by addition of 100 microM thiamine pyrophosphate to buffers used in the purification. The final enzyme preparation was more than 97% pure, as determined by denaturing gel electrophoresis and densitometry. The mobility of the enzyme on a gel filtration column indicates that it is a tetramer of 57-kDa subunits. Large, single crystals of benzoylformate decarboxylase were grown from solutions of buffered polyethylene glycol 400, pH 8.5. The crystals diffract to beyond 1.6 A resolution and are stable for days to X-ray radiation. Analysis of X-ray data from the crystals, along with the newly determined quaternary structure, identifies the space group as I222. The unit cell dimensions are a = 82 A, b = 97 A, c = 138 A. An average Vm value for the crystals is consistent with one subunit per asymmetric unit. The subunits of the tetramer must be arranged with tetrahedral 222 symmetry.     

Increasing the diffraction limit and internal order of a membrane protein crystal by dehydration

It is notoriously difficult to produce crystals of membrane proteins that diffract to sufficient resolution for structural studies by X-ray crystallography. Crystals of a prokaryotic CLC chloride channel that were initially unacceptable for structural analysis improved in both quality and diffraction limit by a process of dehydration. The loss of water decreased the dimensions of the unit cell axes by up to 25 A, improved the diffraction limit from 8.0 to 4.0 A, and decreased the mosaicity to values of approximately 1 degrees. Dehydration of integral membrane protein crystals should be one of the procedures included in the initial screening for appropriate crystals and as a method of improving the diffraction limits of existing crystals.     

Surface crystallisation of the plasma membrane H+-ATPase on a carbon support film for electron crystallography

Large two-dimensional crystals of H+-ATPase, a 100 kDa integral membrane protein, were grown directly onto the carbon surface of an electron microscope grid. This procedure prevented the fragmentation that is normally observed upon transfer of the crystals from the air-water interface to a continuous carbon support film. Crystals grown by this method measure approximately 5 microm across and have a thickness of approximately 240 A. They are of better quality than the monolayers previously obtained at the air-water interface, yielding structure factors to at least 8 A in-plane resolution by electron image processing. Unlike most other two-dimensional crystals of membrane proteins they do not contain a lipid bilayer, but consist of detergent-protein micelles of H+-ATPase hexamers tightly packed on a trigonal lattice. The crystals belong to the two-sided plane group p321 (a=b=165 A), containing two layers of hexamers related by an in-plane axis of 2-fold symmetry. The protein is in contact with the carbon surface through its large, hydrophilic 70 kDa cytoplasmic portion, yet due to the presence of detergent in the crystallizing buffer, the hydrophobicity of the carbon surface does not appear to affect crystal formation. Surface crystallisation may be a useful method for other proteins which form fragile two-dimensional crystals, in particular if conditions for obtaining three-dimensional crystals are known, but their quality or stability is insufficient for X-ray structure determination.     

Crystallization and preliminary X-ray data of bromoperoxidase from Streptomyces aureofaciens ATCC 10762

Bromoperoxidase from Streptomyces aureofaciens ATCC 10762, a non-haem haloperoxidase, has been crystallized using the hanging drop method. Preliminary X-ray diffraction studies show that the crystals belong to the cubic space group P2(1)3 with a = 123.4 A. The asymmetric unit contains a dimer of Mr = 60,200. The crystals diffract to at least 2.3 A resolution and are suitable for crystallographic structure analysis.