Two-dimensional crystallization of a membrane protein on a detergent-resistant lipid monolayer

Two-dimensional crystals of a membrane protein, the proton ATPase from plant plasma membranes, have been obtained by a new strategy based on the use of functionalized, fluorinated lipids spread at the air-water interface. Monolayers of the fluorinated lipids are stable even in the presence of high concentrations of various detergents as was established by ellipsometry measurements. A nickel functionalized fluorinated lipid was spread into a monolayer at the air-water interface. The overexpressed His-tagged ATPase solubilized by detergents was added to the subphase. 2D crystals of the membrane protein, embedded in a lipid bilayer, formed as the detergent was removed by adsorption. Electron microscopy indicated that the 2D crystals were single layers with dimensions of 10 microm or more. Image processing yielded a projection map at 9 A resolution, showing three well-separated domains of the membrane-embedded proton ATPase.     

A simple strategy towards membrane protein purification and crystallization

A simple and cost-efficient detergent screening strategy has been developed, by which a number of detergents were screened for their efficiency to extract and purify the recombinant ammonium/ammonia channel, AmtB, from Escherichia coli, hence selecting the most efficient detergents prior to large-scale protein production and crystallization. The method requires 1 ml cell culture and is a combination of immobilized metal ion affinity chromatography and filtration steps in 96-well plates. Large-scale protein purification and subsequent crystallization screening resulted in AmtB crystals diffracting to low resolution with three detergents. This strategy allows exclusion of detergents with the lowest probability in yielding protein crystals and selecting those with higher probability, hence, reducing the number of detergents to be screened prior to large-scale membrane protein purification and perhaps also crystallization.     

Three-dimensional crystallization of the Escherichia coli glycerol-3-phosphate transporter: a member of the major facilitator superfamily

Here we report the successful three-dimensional crystallization of GlpT, the glycerol-3-phosphate transporter from Escherichia coli inner membrane. GlpT possesses 12 transmembrane alpha-helices and is a member of the major facilitator superfamily. It mediates the exchange of glycerol-3-phosphate for inorganic phosphate across the membrane. Approximately 20 phospholipid molecules per protein, identified as negatively charged phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, were required for the monodispersity of purified GlpT. Analytical size-exclusion chromatography proved to be efficient in identifying detergents for GlpT monodispersity. Nine such detergents were later used for GlpT crystallization. Screening for crystal nucleation was carried out with a variety of polyethylene glycols as the precipitant over a wide pH range. Subsequent identification of a rigid protein core by limited proteolysis and mass spectroscopy resulted in better-ordered crystals. These crystals exhibited order to 3.7 A resolution in two dimensions. However, the stacking in the third dimension was partially disordered. This stacking problem was overcome by using a detergent mixture and manipulating the ionic interactions in the crystallization solution. The resulting GlpT crystals diffracted isotropically to 3.3 A resolution and were suitable for structure determination by X-ray crystallography.     

Crystallization of phycoerythrin 545 of Rhodomonas lens using detergents and unusual additives.

Phycoerythrin 545 from the cryptomonad alga, Rhodomonas lens, has been crystallized under a wide variety of conditions. Although this type of photosynthetic light-harvesting protein is water soluble, detergents were always required for crystallization. The crystals were typically poorly ordered, or ordered in only two dimensions. However, crystals that were well-ordered in three dimensions could be obtained under two different conditions. Both used polyethylene glycol as precipitant and the detergent lauryldimethylaminoxide, but the additives that were critical for obtaining well-ordered crystals were propionamide in one case and Cs+/Br- in the other. Crystals obtained in the presence of propionamide have the space group P2(1)2(1)2(1), with cell constants of a = 85.6 angstroms, b = 108.2 angstroms, and c = 131.0 angstroms, and contain two dimers [i.e., 2 x (alpha2beta2)] in the asymmetric unit. They show diffraction to at least 3.0 angstroms resolution. The crystals grown with Cs+/Br- are nearly isomorphous. Both types of crystals show intense, strongly polarized fluorescence, suggesting that energy transfer in the crystals is highly efficient. This should provide a basis for quantitative investigation of the role of exciton interactions in energy transfer in cryptomonad phycobiliproteins.     

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.     

Novel crystalline sheets of Na,K-ATPase induced by phospholipase A2

Treatment of purified preparations of Na,K-ATPase by phospholipase A2 has led to the formation of two-dimensional crystals of the protein. Control tests with another phospholipase and two detergents have shown that crystallization occurs as the result of hydrolysis and/or solubilization of the phospholipids in the enzyme vesicles. Experimentation with various buffer systems has indicated that reduction in the amount of phospholipids alone is sufficient for inducing the formation of crystalline sheets. Inclusion of crystal inducing ions in the buffer facilitates the crystallization process, resulting in more extensive arrays. The new crystalline sheets are exclusively dimeric with average unit cell dimensions: a = 15.8 +/- 0.4 nm, b = 4.9 +/- 0.2 nm, and gamma = 64 +/- 3 degrees. Examination of the micrographs shows that the initial intermolecular interaction leading to the formation of sheets is between the alpha subunits. Results from this study suggest that removal and/or modification of phospholipids by phospholipases could prove successful in crystallizing those membrane proteins in which excess lipid is the main barrier to the formation of two-dimensional arrays.     

Crystallization of intact monoclonal antibodies

Attempts were made to crystallize four monoclonal antibodies, one IgG_2ak and three IgG_1k. Using a PEG 3350 screen combined with detergents, and developed from our experiments with an IgG_2ak antibody specific for canine lymphoma cells,^1,2 crystals have now been obtained of two of these four immunoglobulins, an antiphenytoin and an antiphenobarbital antibody. A complex between the antiphenobarbital antibody and its drug antigen crystallized as well. The antibody for phenytoin has, to this point, produced only clustered microcrystals, marginally suitable for X-ray analysis. Single crystals of the IgG_1k antibody against phenobarbital, however, were characterized by X-ray diffraction to be primitive monoclinic, with unit cell dimensions a = 67 Å, b = 193 Å, c = 74 Å, and β = 110°. These crystals have an entire IgG_1k molecule as the asymmetric unit and they diffract to at least 3.2 Å resolution. © 1995 Wiley-Liss, Inc.     

Importance of detergent and phospholipid in the crystallization of the human erythrocyte anion-exchanger membrane domain

Three-dimensional crystals were obtained for the membrane domain of the human erythrocyte anion exchanger (AE1, Band 3). Protein homogeneity and stability and the delicate balance between the detergent used and the amount of phospholipids copurifying are critical to the formation of three-dimensional crystals of the AE1 membrane domain. While deglycosylation improved the protein homogeneity, its stability was significantly increased by inhibitor binding. Size-exclusion chromatography showed that the protein was monodisperse in detergents with acyl chains of 10-12 carbons over a pH range of 5.5-10.0. This pH range and the detergents that retained the protein's monodispersity were used for crystallization screening. Crystals were obtained with the protein purified in C(12)E(8), dodecylmaltoside, decylthiomaltoside, and cyclohexyl-hexylmaltoside. Five to 13 lipid molecules per protein were required for the protein crystal formation. Those crystals grown in dodecylmaltoside diffracted X-rays to 14 A. With these factors taken into consideration, ways to further improve the crystal quality are suggested.     

An experiment regarding crystallization of soluble proteins in the presence of beta-octyl glucoside

Twenty-one soluble proteins, five tRNAs, and three protein-nucleic acid complexes were studied in a systematic manner with regard to their crystallization behavior from polyethylene glycol and ammonium sulfate solutions in the presence of 0 to 1.5% beta-octyl glucoside. Our observations suggest that this neutral detergent does influence in a very positive way the growth characteristics of the macromolecules included in this experiment. In general, more reproducible and rapid growth was noted with an increased number of large individual crystals at the expense of microcrystals. In several cases, new crystal forms were discovered. Selected x-ray diffraction analyses imply that crystals grown in the presence of beta-octyl glucoside diffract as well or better than those grown in its absence. In addition, a screen of two proteins grown in the presence of 14 different common detergents suggested that a general detergent effect may be beneficial for the growth of crystals of biological macromolecules.     

Crystallization and preliminary X-ray study of horse pancreatic lipase

Horse (Equus caballus) pancreatic lipase (EC 3.1.1.3) has been crystallized using the hanging drop method of vapour diffusion at 20 degrees C. The best crystals were grown from an 8 mg/ml solution in 10 to 20% (w/v) polyethylene glycol 8000, 10 mM-MgCl2, 0.1 M-NaCl, 0.1 M-Mes buffer (pH 5.6). They reach dimensions of 0.8 mm x 0.4 mm x 0.6 mm. X-ray examination of the lipase crystals shows that they are orthorombic with a space group P2(1)2(1)2(1). Their cell dimensions are a = 79.8 A, b = 97.2 A c = 145.3 A. Two molecules per asymmetric unit give a Vm value of 2.82 A3/dalton (56% water content). Lipase crystals strongly diffract to at least 1.8 A resolution. Some molecular properties of horse lipase compared to those of the better-known porcine enzyme are also presented.     

Characterization of a membrane-associated trimeric low-pH-induced Form of the class II viral fusion protein E from tick-borne encephalitis virus and its crystallization

The interaction of a dimeric membrane anchor-free form of the envelope protein E (sE dimer) from tick-borne encephalitis virus with liposomes at acidic pH levels leads to its conversion into membrane-inserted sE trimers. Electron microscopy shows that these trimers have their long dimensions along the threefold molecular axis, which is oriented perpendicularly to the plane of the membrane, where the protein inserts via the internal fusion peptide. Liposomes containing sE at their surface display paracrystalline arrays of protein in a closely packing arrangement in which each trimer is surrounded by six others, suggesting cooperativity in the insertion process. sE trimers, solubilized with nonionic detergents, yielded three-dimensional crystals suitable for X-ray diffraction analysis.     

Electron microscope observations on Ca2+-ATPase microcrystals in detergent-solubilized sarcoplasmic reticulum

Crystalline arrays of Ca2+-ATPase molecules develop in detergent-solubilized sarcoplasmic reticulum during incubation for several weeks at 2 degrees C under nitrogen in a medium of 0.1 M KCl, 10 mM K-3-(N-morpholino)propanesulfonate, pH 6.0, 3 mM MgCl2, 20 mM CaCl2, 20% glycerol, 3 mM NaN3, 5 mM dithiothreitol, 25 IU/ml Trasylol, 2 micrograms/ml 1,6-di-tert-butyl-p-cresol, 2 mg/ml protein, and 2-4 mg of detergent/mg of protein. Electron microscopy of sectioned, negatively stained, freeze-fractured, and frozen-hydrated Ca2+-ATPase crystals indicates that they consist of stacked lamellar arrays of Ca2+-ATPase molecules. Prominent periodicities of ATPase molecules within the lamellae arise from a centered rectangular lattice of dimensions 164 x 55.5 A. The association of lamellae into three-dimensional stacks is assumed to involve interactions between the exposed hydrophilic headgroups of ATPase molecules, that is promoted by glycerol and 20 mM Ca2+. Similar Ca2+-induced crystals were observed with purified or purified and delipidated Ca2+-ATPase preparations at lower detergent/protein ratios. Cross-linking of Ca2+-ATPase crystals with glutaraldehyde protects the structure against conditions such as low Ca2+, high pH, elevated temperature, SH group reagents, high concentration of detergents, and removal of phospholipids by extraction with organic solvents that disrupt unfixed preparations.     

Capsid-like arrays in crystals of chimpanzee adenovirus hexon

The major coat protein, hexon, from a chimpanzee adenovirus (AdC68) is of interest as a target for vaccine vector modification. AdC68 hexon has been crystallized in the orthorhombic space group C222 with unit cell dimensions of a = 90.8 A, b = 433.0 A, c = 159.3 A, and one trimer (3 x 104,942 Da) in the asymmetric unit. The crystals diffract to 2.1 A resolution. Initial studies reveal that the molecular arrangement is quite unlike that in hexon crystals for human adenovirus. In the AdC68 crystals, hexon trimers are parallel and pack closely in two-dimensional continuous arrays similar to those formed on electron microscope grids. The AdC68 crystals are the first in which adenovirus hexon has molecular interactions that mimic those used in constructing the viral capsid.     

Crystallization and preliminary X-ray diffraction studies of a 40 kDa calcium binding protein specifically expressed in plasmodia of Physarum polycephalum.

A calcium binding protein with a molecular mass of 40 kDa (CBP40), the gene product of plasmodial-specific LAV1-2 of Physarum polycephalum, was crystallized in the presence of EDTA. The crystals diffracted X-rays up to a resolution of 3.0 A. They belonged to the trigonal space group, P3221 (or P3121), with unit cell dimensions of a = b = 64.4 A and c = 207.2 A. Ca2+-bound crystals were obtained by soaking in a CaCl2 solution, which gave diffraction data of similar quality. The Ca2+-soaked crystals belonged to the same space group as those crystallized in the presence of EDTA with unit cell dimensions of a = b = 64.4 A and c = 209.4 A.     

Crystallization of allosteric L-lactate dehydrogenase from Thermus caldophilus and preliminary crystallographic data

L-Lactate dehydrogenase of Thermus caldophilus GK24 was purified from Escherichia coli containing an overexpression plasmid. The enzyme was crystallized from polyethylene glycol 6000 solutions without ligands by the hanging drop vapor diffusion method. Two forms of crystals were obtained. The crystals grown at pH 6.0 were characterized by means of an X-ray diffraction experiment, while those grown at pH 6.5 and 7.0 did not give detectable diffraction spots. The crystals grown at pH 6.0 belonged to monoclinic space group P2(1), the cell dimensions being a = 54.8 A, b = 138.2A, c = 86.1 A, and beta = 93.3 degrees. These crystals diffract to beyond 2.5 A spacing and are stable on X-ray irradiation.     

Structure of rhomboid protease in a lipid environment

Structures of the prokaryotic homologue of rhomboid proteases reveal a core of six transmembrane helices, with the active-site residues residing in a hydrophilic cavity. The native environment of rhomboid protease is a lipid bilayer, yet all the structures determined thus far are in a nonnative detergent environment. There remains a possibility of structural artefacts arising from the use of detergents. In an attempt to address the effect of detergents on the structure of rhomboid protease, crystals of GlpG, an Escherichia coli rhomboid protease in a lipid environment, were obtained using two alternative approaches. The structure of GlpG refined to 1. 7-Å resolution was obtained from crystals grown in the presence of lipid bicelles. This structure reveals well-ordered and partly ordered lipid molecules forming an annulus around the protein. Lipid molecules adapt to the surface features of protein and arrange such that they match the hydrophobic thickness of GlpG. Virtually identical two-dimensional crystals were also obtained after detergent removal by dialysis. A comparison of an equivalent structure determined in a completely delipidated detergent environment provides insights on how detergent substitutes for lipid. A detergent molecule is also observed close to the active site, helping to postulate a model for substrate binding and hydrolysis in rhomboids.     

Projection structure of the bacterial oxalate transporter OxlT at 3.4A resolution

OxlT is a bacterial transporter protein with 12 transmembrane segments that belongs to the Major Facilitator Superfamily of transporters. It facilitates the exchange of oxalate and formate across the membrane of the Gram-negative bacterium Oxalobacter formigenes. From an electron crystallographic analysis of two-dimensional, tube-like crystals of OxlT, we have previously determined the three-dimensional structure of this transporter at 6.5 A resolution. Here, we report conditions to obtain crystalline, two-dimensional sheets of OxlT with diameters exceeding 2 microm. Images of the crystalline sheets were recorded at liquid nitrogen temperatures on a transmission electron microscope equipped with a field-emission gun, operated at 300 kV. Computed optical diffraction patterns from the best images display measurable reflections to about 3.4A, and electron diffraction patterns show spots to about 3.2 A resolution in the best cases. As in the case of the tube-like crystals, the new crystalline sheets also belong to the p22(1)2(1) symmetry group. However, the unit cell dimensions of 102.7A x 67.3 A are significantly smaller in one direction than those previously observed with the tube-like crystals that display unit cell dimensions of 100.3A x 79.0 A. Different regions of OxlT are involved in intermolecular contacts in the two types of crystals, and the improved resolution of the sheet crystals appears to be mainly attributable to this tighter packing of the monomers within the unit cell.     

Crystallization, preliminary X-ray study and crystal activity of the hydrogenase from Desulfovibrio gigas

Hydrogenase (EC 1.12) from Desulfovibrio gigas is a dimeric enzyme (26 and 62 (X 10(3) Mr) that catalyzes the reversible oxidation of molecular hydrogen. Single crystals of hydrogenase have been produced using the hanging drop method, with either PEG (polyethylene glycol) 6000 or ammonium sulfate as precipitants at pH 6.5. X-ray examination of the crystals indicates that those obtained with ammonium sulfate are suitable for structure determination to at least 3.0 A resolution when synchrotron radiation Sources are used (1 A = 0.1 nm). The crystals are monoclinic, with space group C2, and cell dimensions a = 257.0 A, b = 184.7 A, c = 148.3 A and beta = 101.3 degrees, and contain between four and ten molecules per asymmetric unit. The enzyme can be reactivated within the crystals under reducing conditions without crystal damage.     

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.     

Preparation of crystals of chicken cytosolic aspartate amino- transferase, suitable for high resolution x-ray structural analysis

The crystals of cytosolic chicken aspartate aminotransferase were grown from polyethylene glycol solutions. Two of the four crystal modifications obtained diffract to 1.8 A resolution. The crystals of the free holoenzyme belong to space group P2(1)2(1)2(1) with unit cell dimensions of a = 56.9, b = 126.9, c = 124.6 A. The crystals of the enzyme-maleate complex belong to the same space group with slightly different unit cell dimensions of a = 56.5, b = 126.1, c = 124.6 A. The influence of ions of several divalent metals, dioxane and non-ionic detergent beta-octylglucoside on crystallization have been investigated. The best crystals were obtained in the presence of Mg2+ ions. These crystals were used for data collection on the diffractometer.