Sequencing and two-dimensional structure prediction of a phospholipase A(2) inhibitor from the serum of the common tiger snake (Notechis scutatus)

The complex between concanavalin A (Con A) and alpha1-2 mannobiose (mannose alpha1-2 mannose) has been refined to 1.2 A resolution. This is the highest resolution structure reported for any sugar-lectin complex. As the native structure of Con A to 0.94 A resolution is already in the database, this gives us a unique opportunity to examine sugar-protein binding at high resolution. These data have allowed us to model a number of hydrogen atoms involved in the binding of the sugar to Con A, using the difference density map to place the hydrogen atoms. This map reveals the presence of the protonated form of Asp208 involved in binding. Asp208 is not protonated in the 0.94 A native structure. Our results clearly show that this residue is protonated and hydrogen bonds to the sugar. The structure accounts for the higher affinity of the alpha1-2 linked sugar when compared to other disaccharides. This structure identifies different interactions to those predicted by previous modelling studies. We believe that the additional data presented here will enable significant improvements to be made to the sugar-protein modelling algorithms.     

High-resolution two-dimensional electrophoresis of plant proteins

A technique for the analysis of plant proteins from seed, leaf, root, and coleoptile tissues by high resolution two-dimensional electrophoresis is described. This technique is based primarily on the procedure of P. O'Farrell (1975, J. Biol. Chem. 250, 4007-4021); however, a number of improvements and simplifications have been introduced. We have found that resolution of polypeptides from a range of plant tissues is improved if the concentrations of nonionic detergent and ampholytes used in the isoelectric focusing (IEF) step are increased to 4 and 5% (w/v), respectively. Further increase in the concentrations of these two components results in gels of decreased resolution and low mechanical strength. We have also found that substitution of n-octyl beta-D-glucopyranoside or 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate for Triton X-100 or Nonidet-P40 in the IEF dimension significantly increases the resolution of polypeptides in these gels. This technique also allows minor polypeptide differences between closely related cultivars of plants to be identified.     

Kinetics of the barotropic ripple (P beta'')/lamellar liquid crystal (L alpha) phase transition in fully hydrated dimyristoylphosphatidylcholine (DMPC) monitored by time-resolved x-ray diffraction.

We present here the first study of the use of a pressure-jump to induce the ripple (P beta')/lamellar liquid crystal (L alpha) phase transition in fully hydrated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The transition was monitored by using time-resolved x-ray diffraction (TRXRD). Applying a pressure-jump from atmospheric to 11.3 MPa (1640 psig, 111.6 atm) in 2.5 s induces the L alpha to P beta' phase transition which takes place in two stages. The lamellar repeat spacing initially increases from a value of 66.0 +/- 0.1 A (n = 4) to a maximum value of 70.3 +/- 0.8 A (n = 4) after 10 s and after a further 100-150 s decreases slightly to 68.5 +/- 0.3 A (n = 4). The reverse transition takes place following a pressure jump in 5.5 s from 11.3 MPa to atmospheric pressure. Again, the transition occurs in two stages with the repeat spacing steadily decreasing from an initial value of 68.5 +/- 0.3 A (n = 3) to a minimum value of 66.6 +/- 0.3 A (n = 3) after 50 s and then increasing by approximately 0.5 A over a period of 100 s. The transition temperature increases linearly with pressure up to 14.1 MPa in accordance with the Clapeyron relation, giving a dT/dP value of 0.285 degrees C/MPa (28.5 degrees C/kbar) and an associated volume change of 40 microliters/g. A dynamic compressibility of 0.13 +/- 0.01 A/MPa has been determined for the L alpha phase. This value is compared with the equilibrium compressibilities of bilayer and nonbilayer phases reported in the literature. The results suggest testable mechanisms for the pressure-induced transition involving changes in periodicity, phase hydration, chain order, and orientation. A more complete understanding of the transition mechanism will require improvement in detector spatial resolution and sensitivity, and data on the pressure sensitivity of phase hydration.     

Crystal structure of D351A and P312A mutant forms of the mammalian sarcoplasmic reticulum Ca(2+) -ATPase reveals key events in phosphorylation and Ca(2+) release

In recent years crystal structures of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a), stabilized in various conformations with nucleotide and phosphate analogs, have been obtained. However, structural analysis of mutant forms would also be valuable to address key mechanistic aspects. We have worked out a procedure for affinity purification of SERCA1a heterologously expressed in yeast cells, producing sufficient amounts for crystallization and biophysical studies. We present here the crystal structures of two mutant forms, D351A and P312A, to address the issue whether the profound functional changes seen for these mutants are caused by major structural changes. We find that the structure of P312A with ADP and AlF(4)(-) bound (3.5-A resolution) and D351A with AMPPCP or ATP bound (3.4- and 3.7-A resolution, respectively) deviate only slightly from the complexes formed with that of wild-type ATPase. ATP affinity of the D351A mutant was very high, whereas the affinity for cytosolic Ca(2+) was similar to that of the wild type. We conclude from an analysis of data that the extraordinary affinity of the D351A mutant for ATP is caused by the electrostatic effects of charge removal and not by a conformational change. P312A exhibits a profound slowing of the Ca(2+)-translocating Ca(2)E1P-->E2P transition, which seems to be due to a stabilization of Ca(2)E1P rather than a destabilization of E2P. This can be accounted for by the strain that the Pro residue induces in the straight M4 helix of the wild type, which is removed upon the replacement of Pro(312) with alanine in P312A.     

Properties of the detergent solubilised cytochrome c oxidase (cytochrome cbb(3)) purified from Pseudomonas stutzeri.

Cytochrome cbb(3) is a cytochrome c-oxidising isoenzyme that belongs to the superfamily of respiratory haem/copper oxidases. We have developed a purification method yielding large amounts of pure cbb(3) complex from the soil bacterium Pseudomonas stutzeri. This cytochrome cbb(3) complex consists of three subunits (ccoNOP) in a 1:1:1 stoichiometry and contains two b-type and three c-type haems. The protein complex behaves as a monomer with an overall molecular weight of 114.0+/-8.9 kDa and a s(0)(20,w) value of 8.9+/-0.3 S as determined by analytical ultracentrifugation. Crystals diffracting to 5.0 A resolution have been grown by the vapour diffusion sitting drop method to an average size of 0.1 x 0.1 x 0.3 mm. This is the first crystallisation report of a (cbb(3))-type oxidase.     

The solution structure of a locked nucleic acid (LNA) hybridized to DNA

LNA (Locked Nucleic Acids) is a novel oligonucleotide analogue containing a conformationally restricted nucleotide with a 2'-O, 4'-C-methylene bridge that induces unprecedented thermal affinities when mixed with complementary single stranded DNA and RNA. We have used two-dimensional 1H NMR spectroscopy obtained at 750 and 500 MHz to determine a high resolution solution structure of an LNA oligonucleotide hybridized to the complementary DNA strand. The determination of the structure was based on a complete relaxation matrix analysis of the NOESY cross peaks followed by restrained molecular dynamics calculations. Forty final structures were generated for the duplex from A-type and B-type dsDNA starting structures. The root-mean-square deviation (RMSD) of the coordinates for the forty structures of the complex was 0.32A. The structures were analysed by use of calculated helix parameters. This showed that the values for rise and buckle in the LNA duplex is markedly different from canonical B-DNA at the modification site. A value of twist similar to A-DNA is also observed at the modification site. The overall length of the helix which is 27.3 A. The average twist over the sequence are 35.9 degrees +/- 0.3 degrees. Consequently, the modification does not cause the helix to unwind. The bis-intercalation of the thiazole orange dye TOTO to the LNA duplex was also investigated by 1H NMR spectroscopy to sense the structural change from the unmodified oligonucleotide. We observed that the bis-intercalation of TOTO is much less favourable in the 5'-CT(L)AG-3' site than in the unmodified 5'-CTAG-3' site. This was related to the change in the base stacking of the LNA duplex compared to the unmodified duplex.     

Refinement of the nickel site structure in Desulfovibrio gigas hydrogenase using range-extended EXAFS spectroscopy

We have reexamined the Ni EXAFS of oxidized, inactive (as-isolated) and H(2) reduced Desulfovibrio gigas hydrogenase. Better spatial resolution was achieved by analyzing the data over a 50% wider k-range than was previously available. A lower k(min) was obtained using the FEFF code for phase shifts and amplitudes. A higher k(max) was obtained by removing an interfering Cu signal from the raw spectra using multiple energy fluorescence detection. The larger k-range allowed us to better resolve the Ni-S bond lengths and to define more accurately the Ni-O and Ni-Fe bond lengths. We find that as-isolated, hydrogenase has two Ni-S bonds at approximately 2.2 A, but also 1-2 Ni-S bonds in the 2.35+/-0.05 A range. A Ni-O interaction is evident at 1.91 A. The as-isolated Ni-Fe distance cannot be unambiguously determined. Upon H(2) reduction, two short Ni-S bonds persist at approximately 2.2 A, but the remaining Ni-S bonds lengthen to 2.47+/-0.05 A. Good simulations are obtained with a Ni-Fe distance at 2.52 A, in agreement with crystal structures of the reduced enzyme. Although not evident in the crystal structures, an improvement in the fit is obtained by inclusion of one Ni-O interaction at 2.03 A. Implications of these distances for the spin-state of H(2) reduced H(2)ase are discussed.     

In situ hybridization in suspension and flow cytometry as a tool for the study of gene expression.

We describe a method to detect mRNA expression using in situ hybridization in suspension and flow cytometry. Our model was glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression in the leukemic cell line K562. A GAPDH cDNA probe was labeled with digoxigenin-11-dUTP and detected using an FITC-labeled anti-digoxigenin antiserum. We obtained good resolution in specific signals against background (GAPDH signal/control plasmid signal ratio +/- SE 3.5 +/- 0.9). The technique was optimized taking into account several hybridization variables, like fixation, hybridization time, effect of blocking agents, and stringency wash variations. This method also allowed us to quantitate the GAPDH RNA copy number/cell using a fluorescence standard; we obtained a figure of about 1200 copies/cell, which is in good agreement with the dot blot hybridization assay result. Flow cytometric analysis of in situ hybridization represents an original method to study gene expression. This technique has the potential to develop into a multiparametric tool for cell biology studies, examining specific mRNA production together with DNA content or membrane molecules expression, and offering the possibility to purify by sorting a cell population expressing a specific mRNA.     

Crystallization and preliminary X-ray diffraction studies of a flavoprotein, FP390, from a luminescent bacterium, Photobacterium phosphoreum.

A flavoprotein, FP390, obtained from a luminescent bacterium, Photobacterium phosphoreum, in the purification of luciferase has been crystallized by the vapor-diffusion procedure. Crystals obtained from polyethylene glycol 4000 solutions, whose X-ray photographs show powder diffraction patterns, were unsuitable for further crystallographic work. However, tetragonal crystals grown from potassium phosphate solution well diffracted X-rays beyond 3 A resolution. The space group of this crystal is P4(1)22 or P4(3)22 with unit-cell dimensions of a = b = 76.8 and c = 241 A. Assuming two or three molecules in an asymmetric unit, the value for the crystal volume per unit molecular mass, Vm, is calculated as 3.3 or 2.2 A3/Da, respectively. A total of 13,555 independent reflections for the native crystal was collected up to 3 A resolution using a Weissenberg camera attached to the synchrotron radiation source, the merging R factor being 0.077 for 79,335 measurements.     

Fluid bilayer structure determination by the combined use of x-ray and neutron diffraction. I. Fluid bilayer models and the limits of resolution.

This is the first in a series of papers concerned with methods for the determination of the structures of fluid phospholipid bilayers in the liquid-crystalline (L alpha) phase. The basic approach is the joint refinement of quasimolecular models (King and White, 1986. Biophys. J. 49:1047-1054) using x-ray and neutron diffraction data. We present here (a) the rationale for quasimolecular models, (b) the nature of the resolution problem for thermally disordered bilayers, and (c) an analysis of the resolution of experiments in which Gaussian functions are used to describe the distribution of submolecular components. We show that multilamellar liquid-crystalline bilayers are best described by the convolution of a perfect lattice function with a thermally disordered bilayer unit cell. Lamellar diffraction measurements on such a system generally yield only 5-10 orders of diffraction data from which transbilayer profiles of the unit cell can be constructed. The canonical resolution of these transbilayer profiles, defined as the Bragg spacing divided by the index of the highest recorded diffraction order, is typically 5-10 A. Using simple model calculations, we show that the canonical resolution is a measure of the widths of the distributions of constituents of the unit cell rather than a measure of the spatial separation of the distributions. The widths provide a measure of the thermal motion of the bilayer constituents which can be described by Gaussian functions. The equilibrium positions of the centers of the distributions can be determined with a precision of 0.1-0.5 A based upon typical experimental errors.     

Structural and conformational analysis of scorpion (Buthus sindicus) hemocyanin using low resolution techniques

Blue oxygen binding protein hemocyanin from scorpion Buthus sindicus has been investigated using low resolution techniques. The native protein is a polymer of eight different types of subunits arranged in four cubic hexameric form (4x6-mers) as previously annotated using a combination of various types of chromatographic and electrophoretic techniques. In addition, both "top face" as well as the "side view" of the native assembly has also been identified from the negatively stained specimens using transmission electron microscopy confirming the overall structural features of arthropodan hemocyanins. These results are also supported from data obtained from another low resolution technique i.e. Small Angle X-ray scattering (SAXS). SAXS results under oxygenated and deoxygenated states represent a validation case for this technique with key conformational changes of Rg 88.0 --> 86.0 A; +/- 1% (Dmax 280.0 --> 290.0 A; +/- 2%), respectively suggesting that the oxygenated hemocyanin is longer then the deoxygenated hemocyanin by almost 2 A;. Likewise, active conformations of the purified structural and functional subunit Bsin1 under oxygenated and deoxygenated states also determined by SAXS measurements revealed a Rg value of 25.2 --> 25.7 A; +/- 1% (Dmax 75.0 --> 75.5 A; +/- 2%), respectively suggesting very little or no contribution of the individual subunit in the overall conformational change in the native assembly during molecular breathing. Preliminary molecular shapes for the oxy-molecules, calculated directly from the scattering profile-alone in a model-independent procedure, superimpose well on other closely related known three-dimensional structures of the same size. Structural and functional aspects of the native as well as purified subunit and the application of these low resolution techniques like transmission electron microscopy and Small Angle X-ray scattering have been discussed.     

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.     

Changes in the sarcoplasmic reticulum membrane profile induced by enzyme phosphorylation to E1 approximately P at 16 A resolution via time-resolved x-ray diffraction.

Time-resolved x-ray diffraction studies of the isolated sarcoplasmic reticulum (SR) membrane have provided the difference electron density profile for the SR membrane for which the Ca2+ ATPase is transiently trapped exclusively in the first phosphorylated intermediate state, E1 approximately P, in absence of detectable enzyme turnover vs. that before ATP-initiated phosphorylation of the enzyme. These diffraction studies, which utilized the flash-photolysis of caged ATP, were performed at temperatures between 0 and -2 degrees C and with a time-resolution of 2-5 s. Analogous time-resolved x-ray diffraction studies of the SR membrane at 7-8 degrees C with a time resolution of 0.2-0.5 s have previously provided the difference electron density profile for the SR membrane for which the Ca2+ ATPase is only predominately in the first phosphorylated intermediate state under conditions of enzyme turnover vs. that before enzyme phosphorylation. The two difference profiles, compared at the same low resolution (approximately 40 A), are qualitatively similar but nevertheless contain some distinctly different features and have therefore been analyzed via a step-function model analysis. This analysis was based on the refined step-function models for the two different electron density profiles obtained independently from x-ray diffraction studies at higher resolution (16-17 A) of the SR membrane before enzyme phosphorylation at 7.5 and -2 degrees C. The step-function model analysis indicated that the low resolution difference profiles derived from both time-resolved x-ray diffraction experiments arise from a net movement of Ca2+ ATPase protein mass from the outer monolayer to the inner monolayer of the SR membrane lipid bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid (1 hour) high performance gel filtration chromatography resolves coexisting simple micelles, mixed micelles, and vesicles in bile

We describe the use and validation of Superose 6, a high performance gel filtration medium for rapid, high resolution separation and sizing of coexisting simple micelles, mixed micelles, and vesicles in bile. We fractionated model biles (1.7-4.2 g/dl total lipid concentration, 0.15 M NaCl) composed of lecithin (L), cholesterol (Ch), and the common bile salt taurocholate (TC) using Superose 6 gel filtration columns (1.0 cm diameter, 30 cm length, 0.5 ml model bile application, 1.0 ml fractions) pre-equilibrated and eluted with 2.5-10.0 mM TC. Lipid particle sizes were determined by quasielastic light scattering and lipid compositions by conventional analyses. In the absence of L and Ch, pure TC "biles" (32.2 mM), when eluted in the presence of 7.5 mM TC, yielded a single peak of particles (mean hydrodynamic radii, Rh values of 11-15 A), consistent with simple TC micelles. Model biles containing L and TC ([L] = 13.8 mM, [TC] = 32.2 mM) were fractionated with baseline resolution into TC-L mixed micelles, (Rh values of 30-40 A) and simple TC micelles. In agreement with the ternary TC-L-H2O phase diagram (Mazer, N. A., et al. 1980. Biochemistry. 19: 601-615), the proportions of simple and mixed micelles were inversely related to L concentrations ([L] = 0-32.2 mM) and correlated positively with eluant TC concentration. Superose 6 gel fractionation of model biles "super-saturated" with Ch (TC:L:Ch molar ratio 27:63:10, total lipid concentration 3 g/dl) yielded high resolution separation of vesicles (Rh value of 320 A) from mixed micelles of TC-L-Ch (Rh values of 40-50 A) and simple TC micelles (Rh values of 11-15 A). At an eluant TC concentration of 7.5 mM, Ch-rich vesicles (Ch/L molar ratio = 1.6) separated that contained 40% of total Ch, 9% of total L, and no TC, accurately reflecting predictions of the quaternary L-Ch-TC-H2O metastable phase diagram (Mazer, N. A., and M. C. Carey. 1983 Biochemistry. 22: 426-442). This suggested that a 7.5 mM TC concentration approximated the intermicellar concentration under the experimental conditions. We also fractionated an identical model bile using conventional Sephacryl S-300, a medium generally used to study model and native biles. Compared with Superose 6, the Sephacryl S-300 column of equivalent size yielded particle separations with lower resolution and speed (30 h v l h).(ABSTRACT TRUNCATED AT 400 WORDS)     

Sampling the conformational space of membrane protein surfaces with the AFM

The atomic force microscope acquires topographs of single native membrane proteins at subnanometer resolution. Owing to the high signal-to-noise ratio, such images allow the conformational space of membrane protein surfaces to be sampled. This is demonstrated by topographs of porin OmpF, aquaporin-Z, and bacteriorhodopsin, all recorded at a lateral resolution of <7 A and a vertical resolution of ~1 A. The amplitudes of the domain movements were estimated from a large number of single molecule topographs and the corresponding energy landscapes calculated. To visualize the motion of protein domains, movies were generated by similarity ranking of the observed protein configurations. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00249-001-0197-8     

Antagonism by theophylline of the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine at the guinea pig ileum

The inhibitory effect of the putative adenosine A2 receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) on acetylcholine release from the stimulated guinea pig ileum preparation and the nature of its antagonism by theophylline were investigated. NECA was shown to inhibit the response of the ileum preparation in a dose-dependent fashion, and an EC50 value of 1.62 X 10(-8) M was determined. This value was comparable with that determined for the A1 receptor agonist N6-R-phenylisopropyladenosine (R-PIA) (2.57 X 10(-8) M) using the same preparation. Competitive antagonism of the inhibitory effect of NECA by theophylline was quantitated and a pA2 value of 5.04 for the methylxanthine was obtained. This value was similar to those obtained previously for R-PIA and adenosine itself and suggests that these nucleosides may be interacting with the same receptor site on myenteric nerve endings. These findings do not permit the designation of the receptor as an A1 or A2 subtype according to current criteria.     

High spatial resolution readout of 3-D metabolic organ structure: an automated, low-temperature redox ratio-scanning instrument

Intraorgan compartmentation of metabolic processes plays an important role in the understanding of the physiological function of the integrated organ under normal as well as under pathological conditions. We describe here a technique by which 3-D information on tissue redox state may be obtained by means of automated scanning of surface fluorescence. The instrument allows for serial scanning of frozen tissue. A typical scan of a tissue volume of 3 X 3 X 2 mm at a linear resolution of 50 micron and a spatial resolution of ca. 3 X 10(-7) ml includes 144,000 single-point measurements of pyridine nucleotide and flavoprotein fluorescence within the tissue block. The scanning process is fully computerized and programs have been developed which allow 2- or 3-D reconstruction of the data in terms of "redox ratio models," exemplified here by a 3-D model of a spreading depression wave in the cerebral cortex of a gerbil.     

New crystal form of recombinant murine interferon-beta

Although we have reported (Matsuda, S., Kawano, G., Itoh, S., Mitsui, Y., and Iitaka, Y. (1986) J. Biol. Chem. 261, 16207-16209) that recombinant murine interferon-beta produced in Escherichia coli was crystallized in an orthorhombic space group C222(1) using polyethyleneglycol 8000 as precipitant, the crystals had an insufficient resolution and a marked tendency for orientational disorder around the c axis. We now report that another form of murine interferon-beta crystals with little disorder was obtained in the presence of dioxane using ammonium sulfate as precipitant. The new crystals belong to a hexagonal space group P6(1) or P6(5) with a = b = 71.4 A and c = 79.6 A having only one murine interferon-beta molecule in an asymmetric unit. The crystals are reasonably stable to x-rays and significantly diffract up to 2.2 A resolution when a synchrotron beam is used.     

Resolution in electron microscope autoradiography. III. Iodine-125, the effect of heavy metal staining, and a reassessment of critical parameters

Resolution for 125I-labeled specimens under electron microscope (EM) autoradiographic conditions was assessed experimentally. With this isotope the size of the silver halide crystal was the most important resolution-limiting factor. Heavy metal staining such as is routinely used in preparing animal tissues for EM autoradiography produced an improvement in resolution of approximately 15-20%. For a 500-1,000-A biological tissue section fixed with OsO4 and stained with uranyl acetate, we obtained resolution (half distance, HD) values of approximately 800 +/- 120 A using Ilford L4 emulsion and 500 +/- 70 A using a Kodak NTE-type emulsion. General aspects of resolution-limiting factors and comparison with 3H and 14C values are discussed.