Non-thermal microwave effects on protein dynamics? An X-ray diffraction study on tetragonal lysozyme crystals.

X-ray diffraction (XRD) was used to investigate the structural and dynamical effects of microwave fields on tetragonal single crystals of hen egg-white lysozyme at a resolution of 2.0 A. Using a modified slab-line waveguide allows on-line XRD to be carried out while the protein crystal is exposed to well defined microwave fields. High microwave power levels mainly lead to increased, but largely recoverable, lattice defects owing to the evaporation of crystal water. At lower microwave power levels, the presence of the microwave field results in localized reproducible changes in the mean-square displacements (B factors). At particular sites, it is found that the B factors even decrease with increasing microwave power. Most of these effects can be explained by a comparison of the data obtained under microwave irradiation with data obtained at elevated temperature which simulate heating owing to microwave absorption by unbound crystal water. The data show no indication of large microwave-driven displacements of structural subunits in the protein that would be expected if microwaves were to be absorbed resonantly by protein vibrations. Rather, the observed changes in the atomic mean-square displacements suggest that if microwaves couple non-thermally to globular proteins at hydration levels at which they still function, their effect on protein dynamics and structure is very small.

Crystallization and preliminary X-ray analysis of the trehalose/maltose ABC transporter MalFGK2 from Thermococcus litoralis.

Trehalose and maltose uptake in the hyperthermophilic archaeon Thermococcus litoralis is mediated by an ABC transport system. The heterotetrameric transport complex MalFGK(2), consisting of two membrane-spanning subunits and two copies of an ATP-binding cassette protein, has been crystallized. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 106.5, b = 150.5, c = 170.1 A, beta = 107.8 degrees. A native data set has been obtained at a resolution of 5 A.

Active transport of an antibiotic rifamycin derivative by the outer-membrane protein FhuA.

BACKGROUND: FhuA, an integral membrane protein of Escherichia coli, actively transports ferrichrome and the structurally related antibiotic albomycin across the outer membrane. The transport is coupled to the proton motive force, which energizes FhuA through the inner-membrane protein TonB. FhuA also transports the semisynthetic rifamycin derivative CGP 4832, although the chemical structure of this antibiotic differs markedly from that of ferric hydroxamates. RESULTS: X-ray crystallography revealed that rifamycin CGP 4832 occupies the same ligand binding site as ferrichrome and albomycin, thus demonstrating a surprising lack of selectivity. However, the binding of rifamycin CGP 4832 is deviant from the complexes of FhuA with hydroxamate-type ligands in that it does not result in the unwinding of the switch helix but only in its destabilization, as reflected by increased B factors. Unwinding of the switch helix is proposed to be required for efficient binding of TonB to FhuA and for coupling the proton motive force of the cytoplasmic membrane with energy-dependent ligand transport. The transport data from cells expressing mutant FhuA proteins indicated conserved structural and mechanistic requirements for the transport of both types of compounds. CONCLUSIONS: We conclude that the binding of rifamycin CGP 4832 destabilizes the switch helix and promotes the formation of a transport-competent FhuA-TonB complex, albeit with lower efficiency than ferrichrome. Active transport of this rifamycin derivative explains the 200-fold increase in potency as compared to rifamycin, which is not a FhuA-specific ligand and permeates across the cell envelope by passive diffusion only.

Purification, crystallization and preliminary crystallographic analysis of the periplasmic binding protein ProX from Escherichia coli.

A periplasmic binding protein (ProX) for the compatible solutes glycine betaine and proline betaine from Escherichia coli was crystallized using the hanging-drop vapour-diffusion method. Crystals were grown using a protein concentration of 10 mg ml(-1) and a precipitant of 26-28% PEG 4000 in 50 mM PIPES pH 6.2-6.4. Native diffraction data to 1.93 A resolution have been obtained from crystals at 290 K. The crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 48.1, b = 55.0, c = 115.7 A, and contain one molecule per asymmetric unit.

Three-dimensional structure of the nonaheme cytochrome c from Desulfovibrio desulfuricans Essex in the Fe(III) state at 1.89 A resolution.

A nine heme group containing cytochrome c isolated from the soluble and membrane fractions of Desulfovibrio desulfuricans Essex, termed nonaheme cytochrome c, was crystallized, and the structure was solved using the multiple wavelength anomalous dispersion (MAD) phasing method. Refinement was carried out to a resolution of 1.89 A, and anisotropic temperature factors were addressed to the iron and sulfur atoms in the model. The structure revealed two cytochrome c(3) like domains with the typical arrangement of four heme centers. Both domains flanked an extra heme buried under the protein surface. This heme is held in position by loop extensions in each of the two domains. Although both the N- and C-terminal tetraheme domains exhibit a fold and heme arrangement very similar to that of cytochrome c(3), they differ considerably in their loop extensions and electrostatic surface. Analysis of the structure provides evidence for a different function of both domains, namely, anchoring the protein in a transmembranous complex with the N-terminal domain and formation of an electron-transfer complex with hydrogenase by the C-terminal domain.

The crystal structure of a liganded trehalose/maltose-binding protein from the hyperthermophilic Archaeon Thermococcus litoralis at 1.85 A.

We report the crystallization and structure determination at 1.85 A of the extracellular, membrane-anchored trehalose/maltose-binding protein (TMBP) in complex with its substrate trehalose. TMBP is the substrate recognition site of the high-affinity trehalose/maltose ABC transporter of the hyperthermophilic Archaeon Thermococcus litoralis. In vivo, this protein is anchored to the membrane, presumably via an N-terminal cysteine lipid modification. The crystallized protein was N-terminally truncated, resulting in a soluble protein exhibiting the same binding characteristics as the wild-type protein. The protein shows the characteristic features of a transport-related, substrate-binding protein and is structurally related to the maltose-binding protein (MBP) of Escherichia coli. It consists of two similar lobes, each formed by a parallel beta-sheet flanked by alpha-helices on both sides. Both are connected by a hinge region consisting of two antiparallel beta-strands and an alpha-helix. As in MBP, the substrate is bound in the cleft between the lobes by hydrogen bonds and hydrophobic interactions. However, compared to maltose binding in MBP, direct hydrogen bonding between the substrate and the protein prevails while apolar contacts are reduced. To elucidate factors contributing to thermostability, we compared TMBP with its mesophilic counterpart MBP and found differences known from similar investigations. Specifically, we find helices that are longer than their structurally equivalent counterparts, and fewer internal cavities.

Chronic psychiatric patients without psychiatric care: a pilot study.

BACKGROUND: The study is based on the hypothesis that in any catchment area there are patients with chronic mental illness who are unknown to a comprehensive psychiatric/psychosocial care system. METHOD: A standardized questionnaire was sent to all general practitioners in a circumscribed catchment area in southwestern Germany in an attempt to identify such a group, to ascertain what the practitioners considered to be the needs of these patients, and to find out why the patients were not receiving specialized psychiatric care. RESULTS: Of 97 general practitioners contacted, 62 returned the questionnaire. Within a study period of 3 months, 89 patients were identified as having a significant psychiatric disorder, of whom 53% were older than 60 years and 15% were schizophrenic. General practitioners most frequently said that provision of specialized psychiatric care was the most pressing need of these patients, followed by a need for psychosocial services. They also said that the major reason patients did not participate in the mental health system was patient refusal of such services. CONCLUSIONS: Having identified the existence of a group of chronic psychiatric patients who are not receiving specialized psychiatric care, further in-depth field studies to pursue some of the issues raised in this pilot study will be necessary to determine whether further efforts to reach psychiatric patients without defined psychiatric care would be worthwhile. These issues include estimates of the prevalence of such patients in a defined population, patients' more specific reasons for refusal of psychiatric care, and the quality of psychiatric care they receive from general practitioners in comparison with patients who receive more conventional psychiatric care.

The x-ray structure of D-amino acid oxidase at very high resolution identifies the chemical mechanism of flavin-dependent substrate dehydrogenation.

Flavin is one of the most versatile redox cofactors in nature and is used by many enzymes to perform a multitude of chemical reactions. d-Amino acid oxidase (DAAO), a member of the flavoprotein oxidase family, is regarded as a key enzyme for the understanding of the mechanism underlying flavin catalysis. The very high-resolution structures of yeast DAAO complexed with d-alanine, d-trifluoroalanine, and l-lactate (1.20, 1.47, and 1.72 A) provide strong evidence for hydride transfer as the mechanism of dehydrogenation. This is inconsistent with the alternative carbanion mechanism originally favored for this type of enzymatic reaction. The step of hydride transfer can proceed without involvement of amino acid functional groups. These structures, together with results from site-directed mutagenesis, point to orbital orientation/steering as the major factor in catalysis. A diatomic species, proposed to be a peroxide, is found at the active center and on the Re-side of the flavin. These results are of general relevance for the mechanisms of flavoproteins and lead to the proposal of a common dehydrogenation mechanism for oxidases and dehydrogenases.

Crystal structure of MalK, the ATPase subunit of the trehalose/maltose ABC transporter of the archaeon Thermococcus litoralis.

The members of the ABC transporter family transport a wide variety of molecules into or out of cells and cellular compartments. Apart from a translocation pore, each member possesses two similar nucleoside triphosphate-binding subunits or domains in order to couple the energy-providing reaction with transport. In the maltose transporter of several Gram-negative bacteria and the archaeon Thermo coccus litoralis, the nucleoside triphosphate-binding subunit contains a C-terminal regulatory domain. A dimer of the subunit is attached cytoplasmically to the translocation pore. Here we report the crystal structure of this dimer showing two bound pyrophosphate molecules at 1.9 A resolution. The dimer forms by association of the ATPase domains, with the two regulatory domains attached at opposite poles. Significant deviation from 2-fold symmetry is seen at the interface of the dimer and in the regions corresponding to those residues known to be in contact with the translocation pore. The structure and its relationship to function are discussed in the light of known mutations from the homologous Escherichia coli and Salmonella typhimurium proteins.

Crystal structure of Omp32, the anion-selective porin from Comamonas acidovorans, in complex with a periplasmic peptide at 2.1 A resolution.

BACKGROUND: Porins provide diffusion channels for salts and small organic molecules in the outer membrane of bacteria. In OmpF from Escherichia coli and related porins, an electrostatic field across the channel and a potential, originating from a surplus of negative charges, create moderate cation selectivity. Here, we investigate the strongly anion-selective porin Omp32 from Comamonas acidovorans, which is closely homologous to the porins of pathogenic Bordetella and Neisseria species. RESULTS: The crystal structure of Omp32 was determined to a resolution of 2.1 A using single isomorphous replacement with anomalous scattering (SIRAS). The porin consists of a 16-stranded beta barrel with eight external loops and seven periplasmic turns. Loops 3 and 8, together with a protrusion located within beta-strand 2, narrow the cross-section of the pore considerably. Arginine residues create a charge filter in the constriction zone and a positive surface potential at the external and periplasmic faces. One sulfate ion was bound to Arg38 in the channel constriction zone. A peptide of 5.8 kDa appeared bound to Omp32 in a 1:1 stoichiometry on the periplasmic side close to the symmetry axis of the trimer. Eight amino acids of this peptide could be identified, revealing specific interactions with beta-strand 1 of the porin. CONCLUSIONS: The Omp32 structure explains the strong anion selectivity of this porin. Selectivity is conferred by a positive potential, which is not attenuated by negative charges inside the channel, and by an extremely narrow constriction zone. Moreover, Omp32 represents the anchor molecule for a peptide which is homologous to proteins that link the outer membrane to the cell wall peptidoglycan.

The crystal structure of the ligand binding module of axonin-1/TAG-1 suggests a zipper mechanism for neural cell adhesion.

We have determined the crystal structure of the ligand binding fragment of the neural cell adhesion molecule axonin-1/TAG-1 comprising the first four immunoglobulin (Ig) domains. The overall structure of axonin-1(Ig1-4) is U-shaped due to contacts between domains 1 and 4 and domains 2 and 3. In the crystals, these molecules are aligned in a string with adjacent molecules oriented in an anti-parallel fashion and their C termini perpendicular to the string. This arrangement suggests that cell adhesion by homophilic axonin-1 interaction occurs by the formation of a linear zipper-like array in which the axonin-1 molecules are alternately provided by the two apposed membranes. In accordance with this model, mutations in a loop critical for the formation of the zipper resulted in the loss of the homophilic binding capacity of axonin-1.

Crystal structure of the antibiotic albomycin in complex with the outer membrane transporter FhuA.

One alternative method for drug delivery involves the use of siderophore-antibiotic conjugates. These compounds represent a specific means by which potent antimicrobial agents, covalently linked to iron-chelating siderophores, can be actively transported across the outer membrane of gram-negative bacteria. These "Trojan Horse" antibiotics may prove useful as an efficient means to combat multi-drug-resistant bacterial infections. Here we present the crystallographic structures of the natural siderophore-antibiotic conjugate albomycin and the siderophore phenylferricrocin, in complex with the active outer membrane transporter FhuA from Escherichia coli. To our knowledge, this represents the first structure of an antibiotic bound to its cognate transporter. Albomycins are broad-host range antibiotics that consist of a hydroxamate-type iron-chelating siderophore, and an antibiotically active, thioribosyl pyrimidine moiety. As observed with other hydroxamate-type siderophores, the three-dimensional structure of albomycin reveals an identical coordination geometry surrounding the ferric iron atom. Unexpectedly, this antibiotic assumes two conformational isomers in the binding site of FhuA, an extended and a compact form. The structural information derived from this study provides novel insights into the diverse array of antibiotic moieties that can be linked to the distal portion of iron-chelating siderophores and offers a structural platform for the rational design of hydroxamate-type siderophore-antibiotic conjugates.

A conserved structural motif for lipopolysaccharide recognition by procaryotic and eucaryotic proteins.

BACKGROUND: Lipopolysaccharide (LPS), a lipoglycan from the outer membrane of Gram-negative bacteria, is an immunomodulatory molecule that stimulates the innate immune response. High levels of LPS cause excessive release of inflammatory mediators and are responsible for the septic shock syndrome. The interaction of LPS with its cognate binding proteins has not, as yet, been structurally elucidated. RESULTS: The X-ray crystallographic structure of LPS in complex with the integral outer membrane protein FhuA from Escherichia coli K-12 is reported. It is in accord with data obtained using mass spectroscopy and nuclear magnetic resonance. Most of the important hydrogen-bonding or electrostatic interactions with LPS are provided by eight positively charged residues of FhuA. Residues in a similar three-dimensional arrangement were searched for in all structurally known proteins using a fast template-matching algorithm, and a subset of four residues was identified that is common to known LPS-binding proteins. CONCLUSIONS: These four residues, three of which form specific interactions with lipid A, appear to provide the structural basis of pattern recognition in the innate immune response. Their arrangement can serve to identify LPS-binding sites on proteins known to interact with LPS, and could serve as a template for molecular modeling of a LPS scavenger designed to reduce the septic shock syndrome.

Crystallization of the human, mitochondrial voltage-dependent anion-selective channel in the presence of phospholipids.

Overexpressed human voltage-dependent anion-selective channel VDAC or porin from mitochondrial outer membranes has been purified to homogeneity. Electron microscopic analysis of VDAC in detergent solution revealed a uniform particle population consisting of porin monomers. After dialysis of detergent-solubilized porin in the presence of dimyristoylphosphatidylcholine at lipid-to-protein ratios between 0.2 and 0.5 (percentage by weight), mostly multilamellar crystals were obtained. Crystals adsorbed to carbon films flattened during negative staining and air-drying and exhibited different structural features due to differences in the vertical stacking of several crystalline layers, each consisting of one membrane bilayer. Adsorbed, frozen-hydrated multilamellar membrane crystals revealed uniform diffraction patterns with sharp diffraction spots extending to 8.2 A. The surface structure of VDAC was reconstructed from freeze-dried and unidirectionally metal-shadowed crystals. Major protein protrusions were observed from two VDAC monomers present in the unit cell. Differences in the surface structural features indicate alternate orientations of VDAC molecules with respect to the lipid bilayer, allowing the simultaneous imaging of both the cytosolic and intramitochondrial surfaces. Each VDAC molecule consists of a pore lumen with a diameter of 17-20 A surrounded by a protein rim of nonuniform height, suggesting an asymmetrical distribution of protein mass around the diffusion channels.

Crystal structure of a phycourobilin-containing phycoerythrin at 1.90-A resolution.

The structure of R-phycoerythrin (R-PE) from the red alga Griffithsia monilis was solved at 1.90-A resolution by molecular replacement, using the atomic coordinates of cyanobacterial phycocyanin from Fremyella diplosiphon as a model. The crystallographic R factor for the final model is 17.5% (Rfree 22.7%) for reflections in the range 100-1.90 A. The model consists of an (alphabeta)2 dimer with an internal noncrystallographic dyad and a fragment of the gamma-polypeptide. The alpha-polypeptide (164 amino acid residues) has two covalently bound phycoerythrobilins at positions alpha82 and alpha139. The beta-polypeptide (177 residues) has two phycoerythrobilins bound to residues beta82 and beta158 and one phycourobilin covalently attached to rings A and D at residues beta50 and beta61, respectively. The electron density of the gamma-polypeptide is mostly averaged out by threefold crystallographic symmetry, but a dipeptide (Gly-Tyr) and one single Tyr could be modeled. These two tyrosine residues of the gamma-polypeptide are in close proximity to the phycoerythrobilins at position beta82 of two symmetry-related beta-polypeptides and are related by the same noncrystallographic dyad as the (alphabeta)2 dimer. Possible energy transfer pathways are discussed briefly.

Site-directed mutagenesis of loop L3 of sucrose porin ScrY leads to changes in substrate selectivity.

The difference in substrate selectivity of the maltodextrin (LamB) and sucrose (ScrY) porins is attributed mainly to differences in loop L3, which is supposed to constrict the lumen of the pores. We show that even a single mutation (D201Y) in loop L3 leads to a narrowing of the substrate range of ScrY to that resembling LamB. In addition, we removed the putative N-terminal coiled-coil structure of ScrY and studied the effect of this deletion on sucrose transport.

Siderophore-mediated iron transport: crystal structure of FhuA with bound lipopolysaccharide.

FhuA, the receptor for ferrichrome-iron in Escherichia coli, is a member of a family of integral outer membrane proteins, which, together with the energy-transducing protein TonB, mediate the active transport of ferric siderophores across the outer membrane of Gram-negative bacteria. The three-dimensional structure of FhuA is presented here in two conformations: with and without ferrichrome-iron at resolutions of 2.7 and 2.5 angstroms, respectively. FhuA is a beta barrel composed of 22 antiparallel beta strands. In contrast to the typical trimeric arrangement found in porins, FhuA is monomeric. Located within the beta barrel is a structurally distinct domain, the "cork," which mainly consists of a four-stranded beta sheet and four short alpha helices. A single lipopolysaccharide molecule is noncovalently associated with the membrane-embedded region of the protein. Upon binding of ferrichrome-iron, conformational changes are transduced to the periplasmic pocket of FhuA, signaling the ligand-loaded status of the receptor. Sequence homologies and mutagenesis data are used to propose a structural mechanism for TonB-dependent siderophore-mediated transport across the outer membrane.

Mass spectrometric mapping of ion channel proteins (porins) and identification of their supramolecular membrane assembly.

Mass spectrometric peptide mapping, particularly by matrix-assisted laser desorption-ionization (MALDI-MS), has recently been shown to be an efficient tool for the primary structure characterization of proteins. In combination with in situ proteolytic digestion of proteins separated by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), mass spectrometric peptide mapping permits identification of proteins from complex mixtures such as cell lysates. In this study we have investigated several ion channel membrane proteins (porins) and their supramolecular assembly in mitochondrial membranes by peptide mapping in solution and upon digestion in the gel matrix. Porins are integral membrane proteins serving as nonspecific diffusion pores or as specific systems for the transport of substrates through bacterial and mitochondrial membranes. The well-characterized porin from Rhodobacter capsulatus (R.c.-porin) has been found to be a native trimeric complex by the crystal structure and was used as a model system in this study. R.c.-porin was characterized by MALDI-MS peptide mapping in solution, and by direct in situ-gel digestion of the trimer. Furthermore, in this study we demonstrate the direct identification of the noncovalent complex between a mitochondrial porin and the adenine nucleotide translocator from rat liver, by MALDI-MS determination of the specific peptides due to both protein sequences in the SDS-PAGE gel band. The combination of native gel electrophoresis and mass spectrometric peptide mapping of the specific gel bands should be developed as a powerful tool for the molecular identification of protein interactions.

Crystal structure of the effector-binding domain of the trehalose-repressor of Escherichia coli, a member of the LacI family, in its complexes with inducer trehalose-6-phosphate and noninducer trehalose.

The crystal structure of the Escherichia coli trehalose repressor (TreR) in a complex with its inducer trehalose-6-phosphate was determined by the method of multiple isomorphous replacement (MIR) at 2.5 A resolution, followed by the structure determination of TreR in a complex with its noninducer trehalose at 3.1 A resolution. The model consists of residues 61 to 315 comprising the effector binding domain, which forms a dimer as in other members of the LacI family. This domain is composed of two similar subdomains each consisting of a central beta-sheet sandwiched between alpha-helices. The effector binding pocket is at the interface of these subdomains. In spite of different physiological functions, the crystal structures of the two complexes of TreR turned out to be virtually identical to each other with the conformation being similar to those of the effector binding domains of the LacI and PurR in complex with their effector molecules. According to the crystal structure, the noninducer trehalose binds to a similar site as the trehalose portion of trehalose-6-phosphate. The binding affinity for the former is lower than for the latter. The noninducer trehalose thus binds competitively to the repressor. Unlike the phosphorylated inducer molecule, it is incapable of blocking the binding of the repressor headpiece to its operator DNA. The ratio of the concentrations of trehalose-6-phosphate and trehalose thus is used to switch between the two alternative metabolic uses of trehalose as an osmoprotectant and as a carbon source.

Crystallization and preliminary X-ray crystallographic studies of the native and chemically modified anion-selective porin from Comamonas acidovorans.

Omp32, the strongly anion-selective porin from Comamonas acidovorans, has been crystallized. Two crystal forms were observed, both of which belong to space group R3, but exhibit different cell dimensions a = b = 106.7, c = 140.6 A (crystal form I) and a = b = 87.1, c = 135.3 A (crystal form II) with one trimer per asymmetric unit. The crystals diffract to 2.2 and 2.3 A resolution, respectively. Omp32 was chemically modified by introducing negative charges through succinylation. The number and positions of the individual modifications were determined using mass spectrometry and X-ray crystallography. Chemically modified porins yielded crystals of a third form, also of space group R3 but with cell constants of a = b = 109.3 and c = 263.2 A (crystal form III), showing a virtually doubled c axis. Crystals of form III diffract to 3.5 A resolution.