Crystallization of the membrane protein hVDAC1 produced in cell-free system.

Structural studies of membrane proteins are in constant evolution with the development of new improvements for their expression, purification, stabilization and crystallization. However, none of these methods still provides a universal approach to solve the structure of membrane proteins. Here we describe the crystallization of the human voltage-dependent anion channel-1 produced by a bacterial cell-free expression system. While VDAC structures have been recently solved, we propose an alternative strategy for producing the recombinant protein, which can be applied to other membrane proteins reluctant to expression, purification and crystallization by classical approaches. Despite a lot of efforts to crystallize a cell-free expressed membrane protein, this study is to our knowledge one of the first reports of a successful crystallization. Focusing on expression in a soluble and functional state, in a detergent environment, is the key to get crystals. Although the diffraction of VDAC crystals is limited, the simplicity and the rapidity to set-up and optimize this technology are drastic advantages in comparison to other methods.

X-ray structure of bacteriorhodopsin at 2.5 angstroms from microcrystals grown in lipidic cubic phases.

Lipidic cubic phases provide a continuous three-dimensional bilayer matrix that facilitates nucleation and growth of bacteriorhodopsin microcrystals. The crystals diffract x-rays isotropically to 2.0 angstroms. The structure of this light-driven proton pump was solved at a resolution of 2.5 angstroms by molecular replacement, using previous results from electron crystallographic studies as a model. The earlier structure was generally confirmed, but several differences were found, including loop conformations and side chain residues. Eight water molecules are now identified experimentally in the proton pathway. These findings reveal the constituents of the proton translocation pathway in the ground state.

High-resolution structures and dynamics of membrane protein--lipid complexes: a critique.

Crystallographic analysis of lipidic components in complex with membrane proteins reveals molecules exhibiting well-ordered polar or hydrophobic moieties in contact with protein. As dynamic methods indicate high exchange rates, the interpretation of structural data requires a detailed knowledge of the specificity, affinity and cooperativity of lipid binding.

Detergent structure in tetragonal crystals of OmpF porin.

BACKGROUND: The high-resolution structures of five porins have been solved by X-ray crystallography including the trigonal crystal form of the trimeric OmpF porin from Escherichia coli. In an accompanying article, the structure of the tetragonal form of OmpF porin is presented. In contrast to the trigonal crystal form, the protein surfaces normally in contact with lipids in the membrane are exposed and interact with amphiphiles in the tetragonal crystal. Thus, the tetragonal form can be used to investigate protein-detergent interactions. RESULTS: Using single-crystal neutron diffraction studies and two different detergents (one of them deuterated in its hydrophobic moiety), details of the amphiphile-protein interactions are revealed. Detergent molecules bind to the so-called hydrophobic zone that surrounds the OmpF porin trimer and which is exposed to lipid in the native environment. The aromatic rings on both sides of the hydrophobic zone coincide with the boundary between non-polar and polar moieties of the detergents. CONCLUSIONS: In the tetragonal crystal form of OmpF porin, the membrane-exposed area is accessible from the aqueous solution. It is coated by a film of detergent molecules, which presumably mimics the interactions of the protein with lipids in the biological membrane. In the trigonal form, protein-protein interactions predominate in the hydrophobic zone. These may reflect the tight interactions between trimers that are observed in the biological membrane.

Crystal structure of Pseudomonas fluorescens 4-hydroxyphenylpyruvate dioxygenase: an enzyme involved in the tyrosine degradation pathway.

BACKGROUND: In plants and photosynthetic bacteria, the tyrosine degradation pathway is crucial because homogentisate, a tyrosine degradation product, is a precursor for the biosynthesis of photosynthetic pigments, such as quinones or tocophenols. Homogentisate biosynthesis includes a decarboxylation step, a dioxygenation and a rearrangement of the pyruvate sidechain. This complex reaction is carried out by a single enzyme, the 4-hydroxyphenylpyruvate dioxygenase (HPPD), a non-heme iron dependent enzyme that is active as a homotetramer in bacteria and as a homodimer in plants. Moreover, in humans, a HPPD deficiency is found to be related to tyrosinemia, a rare hereditary disorder of tyrosine catabolism. RESULTS: We report here the crystal structure of Pseudomonas fluorescens HPPD refined to 2.4 A resolution (Rfree 27.6%; R factor 21.9%). The general topology of the protein comprises two barrel-shaped domains and is similar to the structures of Pseudomonas 2,3-dihydroxybiphenyl dioxygenase (DHBD) and Pseudomonas putida catechol 2,3-dioxygenase (MPC). Each structural domain contains two repeated betaalpha betabeta betaalpha modules. There is one non-heme iron atom per monomer liganded to the sidechains of His161, His240, Glu322 and one acetate molecule. CONCLUSIONS: The analysis of the HPPD structure and its superposition with the structures of DHBD and MPC highlight some important differences in the active sites of these enzymes. These comparisons also suggest that the pyruvate part of the HPPD substrate (4-hydroxyphenylpyruvate) and the O2 molecule would occupy the three free coordination sites of the catalytic iron atom. This substrate-enzyme model will aid the design of new inhibitors of the homogentisate biosynthesis reaction.

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.

Lipidic cubic phase crystallization of bacteriorhodopsin and cryotrapping of intermediates: towards resolving a revolving photocycle.

Bacteriorhodopsin is a small retinal protein found in the membrane of the halophilic bacterium Halobacterium salinarum, whose function is to pump protons across the cell membrane against an electrostatic potential, thus converting light into a proton-motive potential needed for the synthesis of ATP. Because of its relative simplicity, exceptional stability and the fundamental importance of vectorial proton pumping, bacteriorhodopsin has become one of the most important model systems in the field of bioenergetics. Recently, a novel methodology to obtain well-diffracting crystals of membrane proteins, utilizing membrane-like bicontinuous lipidic cubic phases, has been introduced, providing X-ray structures of bacteriorhodopsin and its photocycle intermediates at ever higher resolution. We describe this methodology, the new insights provided by the higher resolution ground state structures, and review the mechanistic implications of the structural intermediates reported to date. A detailed understanding of the mechanism of vectorial proton transport across the membrane is thus emerging, helping to elucidate a number of fundamental issues in bioenergetics.

Structural basis for lipid-mediated interactions between mitochondrial ADP/ATP carrier monomers.

The oligomerization state of the ADP/ATP carrier is an important issue in understanding the mechanism underlying nucleotide exchange across the inner mitochondrial membrane. The first high resolution structure obtained in the presence of carboxyatractyloside revealed a large cavity formed within a monomer in which the inhibitor is strongly bound. Whereas the protein-protein interactions implicated in the first crystal form are not biologically relevant, the new crystal form described herein, highlights favorable protein-protein interactions. The interactions are mediated by endogenous cardiolipins, which are tightly bound to the protein, two cardiolipins being sandwiched between the monomers on the matrix side. The putative dimerization interface evidenced here is consistent with other structural, biochemical or functional data published so far.

Crystallographic data for the 9000 dalton wheat non-specific phospholipid transfer protein.

The wheat non-specific phospholipid transfer protein belongs to a family of small proteins sharing a common pattern of four disulphide bridges. Its function in vivo is not known, but it has a high affinity to phospholipids and is involved in phospholipid transfer in vitro. The molecular weight is 9607, and it crystallizes in the space group P2(1) with a = 40.73 A, b = 112.11 A, c = 50.44 A and beta = 106.80 degrees. The crystals diffract to 3 A resolution.

Crystallographic data for soybean hydrophobic protein.

The soybean hydrophobic protein belongs to a family of proteins that contains a number of storage and phospholipid binding proteins. Its function is not known, but its overall hydrophobic nature is typical of many membrane proteins of similar size. The molecular weight is 8.3 x 10(3), and it crystallizes in the space group P2(1)2(1)2(1), with a = 52.01 A, b = 43.50 A and c = 28.80 A. The crystals diffract to 1.8 A resolution, and are thus suitable for X-ray structural studies.

Crystallization and preliminary crystallographic data for acetohydroxy acid isomeroreductase from Spinacia oleracea.

Acetohydroxy acid isomeroreductase (EC 1.1.1.86) is one of the enzymes involved in branched-chain amino acid biosynthesis. The enzyme from spinach (Spinacia oleracea) leaves has been crystallized using the hanging drop vapour diffusion method. The free enzyme crystallized from polymethylene glycol solutions, but these crystals were unsuitable for X-ray diffraction analysis. In the presence of NADPH, Mg(2+) and a reaction intermediate analogue (2-dimethylphosphinoyl-2-hydroxy acetic acid (Hoe 704) or N-hydroxy-N-isopropyloxamate (IpOHA)), much better crystals were obtained. Crystals grown from ammonium sulphate belong to space group P2(1) with cell dimensions a + 193.78(7) A, b = 63.69(2) A, c = 112.84(1) A and beta = 121.22(1) degrees. The molecular mass of the protein, the volume of the unit cell, and crystal density measurements indicated that the asymmetric unit contains two dimers. X-ray diffraction patterns showed measurable reflections to beyond 2.5 A.

Crystal structure of hydrophobic protein from soybean; a member of a new cysteine-rich family.

X-ray diffraction methods have been used to determine the structure of the 8.3 kDa hydrophobic protein from soybean and to refine the atomic co-ordinates to a crystallographic R-factor of 18.7% at 1.8 A resolution. The molecule is a four-helix bundle, which together with the connecting loops and a twisted beta-strand form a spiral. The surface contains 70% apolar atoms, and the crystal packing is dominated by hydrophobic interactions, producing a two-dimensional sheet of protein molecules. Most of the 59 water molecules located are involved in hydrophilic contacts and their structural organization does not seem to be affected by the high hydrophobicity of the molecule. From the protein fold it appears that three of the four disulphide bridges are important for keeping the amino and carboxyl-terminal segments in place in the native form, while the central part of the molecule is stabilized by many hydrophobic interactions. Although the protein function is not known, a number of possibilities can be excluded on experimental grounds and by comparison with other members of the family.

Detergent-free membrane protein crystallization.

A comprehensive understanding of structure-function relationships of proteins requires their structures to be elucidated to high resolution. With most membrane proteins this has not been accomplished so far, mainly because of their notoriously poor crystallizability. Here we present a completely detergent-free procedure for the incorporation of a native purple membrane into a monoolein-based lipidic cubic phase, and subsequent crystallization of three-dimensional bacteriorhodopsin crystals therein. These crystals exhibit comparable X-ray diffraction quality and mosaicity, and identical crystal habit and space group to those of bacteriorhodopsin crystals that are grown from detergent-solubilized protein in cubic phase.

Complete amino acid sequence of puroindoline, a new basic and cystine-rich protein with a unique tryptophan-rich domain, isolated from wheat endosperm by Triton X-114 phase partitioning.

A new basic protein has been isolated from wheat endosperm by Triton X-114 phase partitioning. It contains five disulfide bridges and is composed of equal amounts of a polypeptide chain of 115 amino acid residues and of the same chain with a C-terminus dipeptide extension. The most striking sequence feature is the presence of a unique tryptophan-rich domain so that this protein isolated from wheat seeds has been named puroindoline. The similar phase partitioning behavior in Triton X-114 of this basic cystine-rich protein and of purothionins suggests that puroindoline may also be a membranotoxin that might play a role in the defense mechanism of plants against microbial pathogens.

Detergent binding in trigonal crystals of OmpF porin from Escherichia coli.

The structure of the detergent, ocytyl hydroxyethylsufoxide (C8(HE)SO), bound to the OmpF porin from E coli (in the trigonal crystal form) has been determined by neutron crystallography. Due to a dynamic exchange of detergent molecules with their environment they are not ordered on an atomic scale. The structure reported here is therefore at a resolution of approximately 16 A. The X-ray crystallographically determined structure of the protein provides a starting point for the neutron analysis in which the detergent is visualized primarily thanks to its high contrast against D2O. The structure shows the detergent to be located mainly in two areas. It forms toroidal annuli around each OmpF trimer, these annuli fusing to form a detergent belt surrounding a solvent filled column traversing the crystal. Those areas of the protein to which the detergent binds are formed almost exclusively of hydrophobic residues and form a band about 30 A high around the trimer. Its upper and lower bounds are defined by two bands of aromatic residues, tyrosines pointing away from the detergent belt and interacting with the polar headgroups while phenylalanines point inwards. This strongly suggests that the same areas define, in vivo, the location at which protein interacts with lipid. The hydrophobic moiety of detergent is also found mediating the hydrophobic protein-protein interactions at the interface between two trimers on the crystallographic two-fold axis.

The interplay between X-ray crystallography, neutron diffraction, image reconstruction, organo-metallic chemistry and biochemistry in structural studies of ribosomes.

Crystals of ribosomes, their complexes with components of protein biosynthesis, their natural, mutated and modified subunits, have been subjected to X-ray and neutron crystallographic analyses. Electron microscopy and 3-dimensional image reconstruction, supported by biochemistry, genetic, functional and organo-metallic studies were employed for facilitating phasing of the crystallographic data. For example, a monofunctional multi heavy-atom cluster (undecagold) was designed for covalent and quantitative binding to ribosomes. The modified particles were crystallized isomorphously with the native ones. Their difference-Patterson maps contain indications for the usefulness of these derivatives for subsequent phasing. Models of the ribosome and its large subunit were reconstructed from tilt series of 2-dimensional sheets. The comparison of the various reconstructed images enabled an initial assessment of the reliability of these models and led to tentative assignments of several functional features. These include the presumed sites for binding mRNA and for codon-anticodon interactions, the path taken by the nascent protein chain and the mode for tRNA binding to ribosomes. These assignments assisted in the design of biologically meaningful crystal systems. The reconstructed models are being used to identify structural features in initial density maps derived from X-ray and neutron diffraction data.

Detergent organisation in solutions and in crystals of membrane proteins.

The use of neutron scattering in studying the organisation of detergents in pure micelles, in protein/detergent mixed micelles and in crystals of membrane proteins, is reviewed. Small angle scattering has been used to study the size, shape and composition of pure and mixed protein/detergent micelles as well as the effects of adding small amphiphiles. The technique of contrast variation applied to single crystals is described and its application to the determination of the organization of detergent in single crystals of membrane proteins is discussed. A better understanding of protein/detergent interactions should help in producing crystals of membrane proteins more easily as well as clues to the nature of protein/lipid interactions in vivo.

Location of diphenyl-hexatriene and trimethylammonium-diphenyl-hexatriene in dipalmitoylphosphatidylcholine bilayers by neutron diffraction.

Neutron scattering experiments have been performed on oriented dipalmitoylphosphatidylcholine (DPPC) bilayers containing diphenylhexatriene (DPH) or its trimethylammonium analog (TMA-DPH). DPH and TMA-DPH were either protonated or deuterated in one of the phenyl rings which afforded by using proton-deuterium contrast methods the location of these fluorescent probes in the model membrane. Both probes exhibit bimodal distributions in DPPC. The position, population and orientation in the two sites vary depending upon the physical state of the bilayer (gel or fluid) and the presence or absence of the TMA group. In gel (L beta') phase lipids DPH is located close and parallel to the bilayer surface (site I) and near the bilayer center, oriented at approximately 30 degrees with respect to the normal to the surface (site II). On going to the fluid (L alpha) phase, a distribution of orientations around the parallel to the surface is only observed for site II. Orientation of DPH in site I is unchanged. In the gel phase TMA-DPH is found in a position close and parallel to the bilayer surface (site I) and in a position (site II) oriented at an angle of approximately 25 degrees with respect to the bilayer normal, with the trimethylammonium group anchored in the head group domain. On going to the fluid phase there is a change in molecular orientation of each of the sites. In site I the molecule penetrates deeper in the bilayer and adopts a approximately 20 degrees tilt with respect to the surface, with an orientational distribution of +/- 10 degrees. In site II the molecule becomes perpendicular to the membrane surface. Changes in population of sites, both with DPH and TMA-DPH, are observed on going from low to high temperatures. They are however difficult to quantitate due to experimental conditions. The H2O-2H2O exchange experiments afforded an estimate of the water layer thickness as well as the maximum penetration of water into the interior of the bilayer.

Spectroscopic characterization of bacteriorhodopsin's L-intermediate in 3D crystals cooled to 170 K.

Spectra are presented from a single 3D microcrystal of bacteriorhodopsin (bR) cooled to 170 K under various illumination conditions. This set is necessary and sufficient to assign the relevant crystal reference spectra. A spectral decomposition of the difference spectrum obtained following the trapping protocol of Royant et al. (2000) (Nature 406, 645-648) is given, confirming that the low temperature L-intermediate was the species that dominated the structural rearrangements previously reported. Smaller contributions from the K and M spectral intermediates are also quantified. Mechanistic insights derived from the X-ray structures of the early bR intermediates are discussed.

Relations between structure and function of the mitochondrial ADP/ATP carrier.

Import and export of metabolites through mitochondrial membranes are vital processes that are highly controlled and regulated at the level of the inner membrane. Proteins of the mitochondrial carrier family ( MCF ) are embedded in this membrane, and each member of the family achieves the selective transport of a specific metabolite. Among these, the ADP/ATP carrier transports ADP into the mitochondrial matrix and exports ATP toward the cytosol after its synthesis. Because of its natural abundance, the ADP/ATP carrier is the best characterized within MCF, and a high-resolution structure of one conformation is known. The overall structure is basket shaped and formed by six transmembrane helices that are not only tilted with respect to the membrane, but three of them are also kinked at the level of prolines. The functional mechanisms, nucleotide recognition, and conformational changes for the transport, suggested from the structure, are discussed along with the large body of biochemical and functional results.