Immunodominant proteins P1 and P40/P90 from human pathogen Mycoplasma pneumoniae.

Mycoplasma pneumoniae is a bacterial human pathogen that causes primary atypical pneumonia. M. pneumoniae motility and infectivity are mediated by the immunodominant proteins P1 and P40/P90, which form a transmembrane adhesion complex. Here we report the structure of P1, determined by X-ray crystallography and cryo-electron microscopy, and the X-ray structure of P40/P90. Contrary to what had been suggested, the binding site for sialic acid was found in P40/P90 and not in P1. Genetic and clinical variability concentrates on the N-terminal domain surfaces of P1 and P40/P90. Polyclonal antibodies generated against the mostly conserved C-terminal domain of P1 inhibited adhesion of M. pneumoniae, and serology assays with sera from infected patients were positive when tested against this C-terminal domain. P40/P90 also showed strong reactivity against human infected sera. The architectural elements determined for P1 and P40/P90 open new possibilities in vaccine development against M. pneumoniae infections.

Production and characterization of recombinant P1 adhesin essential for adhesion, gliding, and antigenic variation in the human pathogenic bacterium, Mycoplasma pneumoniae.

Mycoplasma pneumoniae forms an attachment organelle at one cell pole, binds to the host cell surface, and glides via a unique mechanism. A 170-kDa protein, P1 adhesin, present on the organelle surface plays a critical role in the binding and gliding process. In this study, we obtained a recombinant P1 adhesin comprising 1476 amino acid residues, excluding the C-terminal domain of 109 amino acids that carried the transmembrane segment, that were fused to additional 17 amino acid residues carrying a hexa-histidine (6 × His) tag using an Escherichia coli expression system. The recombinant protein showed solubility, and chirality in circular dichroism (CD). The results of analytical gel filtration, ultracentrifugation, negative-staining electron microscopy, and small-angle X-ray scattering (SAXS) showed that the recombinant protein exists in a monomeric form with a uniformly folded structure. SAXS analysis suggested the presence of a compact and ellipsoidal structure rather than random or molten globule-like conformation. Structure model based on SAXS results fitted well with the corresponding structure obtained with cryo-electron tomography from a closely related species, M. genitalium. This recombinant protein may be useful for structural and functional studies as well as for the preparation of antibodies for medical applications.

Characterization of the binding of HU and IHF, homologous histone-like proteins of Escherichia coli, to curved and uncurved DNA.

The binding of E. coli histone-like protein HU to curved and uncurved DNA fragments containing adenine tracts was characterized by relative binding affinity assay, and compared with that of other homologous histone-like protein integration host factor (IHF). Both HU and IHF have about 3- to 5-fold higher affinity for overall curved DNA fragments such as (A6N4)11 and (A3T3N4)12 compared to a standard duplex fragment with mixed sequence. The binding manner of HU to the curved fragments was highly cooperative. However, loss of overall curvature for shorter fragments (< approximately 100 bp) reduced the preference of HU binding to curved (A3T3N4)n over uncurved (T3A3N4)n, indicating that the binding specificity of HU to curved DNA is length-dependent. Thus, the curved DNA configuration of the whole molecule facilitates the binding of several HU molecules to form the hierarchy of HU-DNA complex. Furthermore, it was shown that HU and IHF bind less well to (A6N9)n, which has a zig-zag straight structure, whereas they preferentially bind to uncurved (T3A3N4)14. These results suggested that not only intrinsically overall curvature but also the preferred orientations for DNA bending in the protein-DNA complex are important factors for affinities of HU and IHF.

The binding specificity and affinity of E. coli integration host factor (IHF) are influenced by the flexibility of flanking regions of its recognition sites.

To understand the roles of the 5'-flanking region of the recognition sites in binding specificity and the affinity of integration host factor (IHF), a variety of DNA fragments with a 13-bp consensus sequence, 5'-WATCAAN4TTR-3'[Friedman, Cell, 55, 545 (1988)], but with different 5'-flanking sequences were investigated by gel retardation and methylation interference assays. It has been well-established that the putative A/T rich element distal from the 5'-end of the consensus made a significant contributions to the binding of IHF. However, many of the DNA fragments used here revealed specific binding to IHF without such an A/T element. Several bases neighboring to the 5'-end of the consensus sequence had significant effects on the binding specificity as well as its affinity, and these results indicate that the sequence-directed bendability of the flanking region plays an important role in the specific recognition by IHF.

Selective separation of human peripheral platelets, granulocytes and lymphocytes by surface affinity chromatography.

Selective separation of human peripheral platelets, granulocytes and lymphocytes was investigated by column liquid chromatography using methoxyethoxymethyl (MEM) bonded-phase columns (25 x 0.9 cm I.D.). Isotonic solutions containing mono- and disaccharides, methyl-alpha-D-pyranosides and a physiological saline at pH 7.4 were used as the mobile phase. Granulocytes and lymphocytes were separated on the MEM-Cellulofine GH-25 column by elution with 0.3 M D-mannose solution. The isolation of platelets and lymphocytes from human leukocyte-rich plasma was performed with a MEM-Sephadex G25 column and elution with 0.27 M sucrose solution. On the same column platelets could also be collected selectively by elution with 0.31 M methyl-alpha-D-mannoside at the high recovery of 100%. The isolated cells were viable for more than 90%.

Complementary use of counter-current chromatography and hydroxyapatite chromatography for the separation of three main classes of lipoproteins from human serum.

High-density, low-density and very-low-density lipoproteins (HDLs, LDLs and VLDLs) were purified from human serum by the combined use of counter-current chromatography (CCC) and hydroxyapatite chromatography. Polymer-phase CCC of human serum using the cross-axis coil planet centrifuge yielded two lipoprotein fractions, one containing HDLs and LDLs and the other VLDLs and serum proteins. Each fraction was concentrated and subjected to hydroxyapatite chromatography to obtain three lipoprotein fractions, all free from serum proteins. Each lipoprotein was confirmed by agarose gel electrophoresis.

The loop sequence plays crucial roles for isomerization of intramolecular DNA triplexes in supercoiled plasmids.

The effect of base composition in the central region of polypurine.polypyrimidine (Pur.Pyr) tracts on the formation of intramolecular DNA triplexes in plasmids was examined using chemical probes (diethyl pyrocarbonate and OsO4), and two-dimensional (2-D) agarose gel electrophoresis. Two isomers exist for an intramolecular triplex: one with the 3'-half of the Pyr strand as the third strand (H-y3) and the other with the 5'-half of the Pyr strand as the third strand (H-y5). It was shown that the content and position of G + C residues in the triplex loop region (the center of Pur.Pyr tracts) are primary determinants for the isomerization between the H-y3 and H-y5 triplexes. Divalent metal ions such as Mg2+ and negative supercoiling also modulate the isomerization: the H-y5 conformation is stabilized by the divalent metal ions and/or under relatively lower negative supercoiling. 2-D gel analyses revealed that two isomers, H-y3 and H-y5, are topologically non-equivalent: the H-y3 formation relaxes one more supercoil turn than H-y5. As the G + C content in the center of Pur.Pyr tracts increases, the triplex requires more supercoil energy for formation. Therefore, the base-pair opening in the center of Pur.Pyr tracts is the initial and critical step in the pathway for the formation of triplex as well as the isomerization. The role of the triplex loop sequence is explained by a model in which the nucleation process of H-y3 formation requires a wide range of base-pair opening compared to that of H-y5: such unwinding would not be favored for the central region of the duplex with high G + C content and so it would be in the presence of Mg2+, and thereby the H-y5 formation is promoted.

Proton NMR study on a histone-like protein, HU alpha, from Escherichia coli and its complex with oligo DNAs.

It was confirmed that the flexible arm region of HU alpha forms an antiparallel beta-sheet and that all of the residues of phenylalanines, together with some of leucines and/or valines, form a hydrophobic core within the dimer of HU alpha. HU alpha protein alone is thermally labile and melts at 38 degrees C, but it becomes remarkably stabilized and melts at 59 degrees C in the presence of DNA. Several resonances from both HU alpha and DNA perturbed by their complex formation, notably those of His C-2 and C-4 protons, downfield shifted C alpha protons in the antiparallel beta-sheet, as well as Arg C delta and Lys C epsilon protons. The results indicated that a beta-sheet region of HU alpha binds to DNA, and also showed that rapid equilibrium occurs on the NMR time scale between bound and unbound states of HU alpha. A few intermolecular nuclear Overhauser effects (NOEs) were also observed between the protein and H1' protons of DNA in the complex, suggesting that HU alpha binds primarily to the minor groove of DNA.

Specific and nonspecific interactions of integration host factor with oligo DNAs as revealed by circular dichroism spectroscopy and filter binding assay.

Binding specificity of integration host factor (IHF) to oligo DNAs has been studied by circular dichroism (CD) spectroscopy and filter binding experiment. CD difference spectra of IHF-DNA complexes demonstrated that a conformational change in DNA was induced by binding of IHF when DNA had a consensus sequence for the binding sites of IHF, but that such conformational change was not observed for consensus DNA 20 mer as well as nonconsensus DNA 45 mer. Dissociation constants for IHF-DNA complexes determined by filter binding assay showed that IHF has indeed stronger affinity to DNA with the consensus binding site than to nonconsensus DNA, but the difference in its affinity between consensus and nonconsensus DNAs was rather small, 3.4-fold. It was, therefore, concluded that the flanking regions of the consensus sequence are important for the specific binding of IHF and that its binding specificity is well characterized by the induced conformational change in DNA rather than by dissociation constants for IHF-DNA complexes.

Separation of human serum lipoproteins into three major classes by hydroxyapatite chromatography.

The separation of normolipidemic male serum lipoprotein fraction, prepared by ultracentrifugal flotation, was studied on hydroxyapatite columns. Potassium phosphate buffers in the pH range 5.6-7.4 were evaluated as eluents. The three main classes of the lipoproteins (high density, low density and very low density) can be separated on the Tiselius-type hydroxyapatite (Bio-Gel HTP DNA grade) column by elution with 75, 250 and 300 mM potassium phosphate buffer (pH 7.4), respectively.

Surface affinity chromatographic separation of blood cells. VII. Relationship between capacity factors of human peripheral blood cells and the rate of penetration of liquids into xerogel column packings.

Eleven kinds of column packing gels which bonded poly(ethylene glycol) (PEG) to Sepharose 6B (PEG-C10-Sepharose) were prepared. Human peripheral blood cells were chromatographed on these gel columns by eluting with 0.09 M phosphate-buffered 2% (w/w) dextran T40 solution at the pH of the respective isoelectric points of the blood cells. The rate of penetration of water or the mobile phases into the PEG-C10-Sepharose xerogels as a measure of the hydrophobicity of the gels depended on both the oxyethylene residue content and the number of oxyethylene units of the packing gels. The capacity factors of granulocytes and lymphocytes were increased on the columns packed with gels having a slower rate of penetration of the liquids into the xerogels.

Circular dichroic investigations into binding of sulfonamides to serum albumin.

The binding of twelve structurally related sulfonamides to serum albumins including human was investigated using a circular dichroic technique. Some differences of circular dichroic spectral characteristics were observed when sulfonamides were bound to the same albumin or when the drug was bound to several albumins. The differences in these circular dichroic characteristics may be due to various asymmetries. The Scatchard plots indicated that only the primary site was capable of inducing ellipticities of the drugs. The interaction with rabbit serum albumin showed significantly large binding constants and apparent anisotropy factors (g' values), in comparison with other albumins. No significant correlation between the g' values of the induced circular dichroic bands and partition coefficients or/and pKa values was observed. The induced ellipticities of the drug-albumin complexes decreased with pH. This pH dependence can be explained by the ionization of drug and albumin as well as the conformational change of the albumin.

Effect of pH and small inorganic ions on binding of sulfadimethoxine and sulfaphenazole to human serum albumin measured by circular dichroism.

The binding of sulfadimethoxine and sulfaphenazole to human serum albumin (HSA) has been shown by circular dichroism measurements to be dependent on the N-B transition. The secondary drug binding sites were found to be optically active in the B conformation form in HSA but optically inactive in the N form. Moreover, the drug-HSA interaction in Tris-HCl buffer seems to be more sensitive to the conformational change in HSA, compared with that in the phosphate buffer.

Spectroscopic study of the interaction of coumarin anticoagulant drugs with polyvinylpyrrolidone.

The interaction of coumarin anticoagulants with polyvinylpyrrolidone (PVP) was investigated using a fluorescence technique. The fluorescence intensities of warfarin and phenprocoumon were greatly enhanced following binding to PVP, while the fluorescence of 4-hydroxycoumarin was little enhanced in the presence of PVP. The enhanced fluorescence of warfarin and phenprocoumon bound to PVP can be explained by their incorporation into the hydrophobic environment in the PVP and by a decrease in the internal rotation of the alpha-substituted benzyl group in the drugs. The binding parameters of warfarin and phenprocoumon were estimated by the Klotz method; the binding constants for phenprocoumon and warfarin were found to be 2.6 X 10(4) and 2.2 X 10(4) M-1, respectively. The 13C-NMR measurements suggest the lactone moiety in the 4-hydroxycoumarin and the substituted benzene ring play an important role in the binding to PVP.

Fluorescence high performance liquid chromatographic analysis of free fatty acid levels changed during aggregation of rat platelets.

Stimuli-induced changes in free fatty acid levels of activated platelets were assayed by reversed-phase high performance liquid chromatography. The fatty acids were prelabeled as their fluorescent 9-aminophenanthrene derivatives for the detection. The levels of saturated fatty acids such as palmitic acid in an extracellular medium of rat platelet-rich plasma are significantly raised by stimulation with 10 microM ADP or 8 micrograms/ml collagen. Similar increases in saturated fatty acids liberated from washed rat platelets are also observed by using thrombin stimulation. Quantitative changes in the intra- and extracellular levels of fatty acids induced by washed platelet aggregation were assayed after addition of thrombin for 15 min. Increase in palmitic and stearic acid was observed approximately 3-fold their levels of that the unstimulated control.

Nuclear magnetic spectra of self complementary decanucleotides in solution; base sequence effect on the chemical shifts of nonexchangeable protons.

The aim of this study was to attempt to determine the extent to which the chemical shifts of the nonexchangeable base protons of a DNA helix depend upon the base sequence. We measured the proton NMR spectra of twelve decadeoxynucleotides in order to carry out a "statistical" treatment. In the helices, the chemical shifts were found to be determined within +/- 0.04 ppm, largely by the nearest neighbor residues on the 5'-side, and to a smaller extent by the residue on the 3'-side. The theoretical chemical shift calculations reproduced very well the polymerization shifts measured for H2 protons of adenosines if the electrostatic field effect was taken into account. A fair agreement was also obtained for H8 protons of the adenosine and guanosine residues. However, theory underestimates the polarization effects of the base protons of cytidine. This discrepancy suggests that the conformation of this residue is different in the mononucleotides relative to double helices.

In vivo phosphorylation of histones H1 and H5 in calf thymus and chicken erythrocyte as studied by 31P nuclear magnetic resonance spectroscopy.

The 31P NMR method was first applied to characterize in vivo phosphorylation of H1 and H5 in calf thymus and chicken erythrocytes as well as in vitro phosphorylation of H1 and H5 by cAMP-dependent protein kinase. The amino acid residues phosphorylated in vivo in the histones were exclusively serine residues, and the mole fraction of phosphoserine was estimated to be 0.34 and 0.27 per molecule of calf thymus H1 and chicken erythrocyte H5, respectively. Interestingly, chicken erythrocyte H1 was not phosphorylated in vivo. Three H1 subtypes from calf thymus H1 varied in the 31P NMR spectra, and the bisected fragments of calf thymus H1 and chicken erythrocyte H5 exhibited characteristic spectral patterns, indicating that there are considerable diversities of the degree of phosphorylation and phosphorylation sites in very-lysine-rich histones. Furthermore, it was found that the microenvironment of phosphoserine residues phosphorylated in vivo in calf thymus H1 and chicken erythrocyte H5 is quite distinct from that of phosphoserine residues phosphorylated in vitro by bovine heart cAMP-dependent protein kinase.

Surface affinity chromatographic separation of blood cells. VI. Capacity factors of human peripheral blood cells on poly(propylene glycol) bonded chromagel columns and their correlation with surface hydrophobicities of the gel beads and the cells.

The chromatographic behaviour of human peripheral blood cells was investigated on poly(propylene glycol) (PPG)-bonded Chromagel. The mobile phase was 0.09 M phosphate-buffered 2% (w/w) dextran T40 solution at the pH of the respective isoelectric points of human blood cells. The elution order of four kinds of blood cells from PPG-C3-Chromagel columns coincided with that of the delta log K values of these cells determined by the hydrophobic affinity partition method using Pluronic P84 as a hydrophobic ligand. The capacity factors of granulocytes and lymphocytes increased with increase in the delta log K values of PPG-C3-Chromagel beads. Hydrophobic interactions contributed to the retention of the four kinds of blood cells on PPG-bonded Chromagel columns.

Tyrosine-specific dephosphorylation-phosphorylation with alkaline phosphatases and epidermal growth factor receptor kinase as evidenced by 31P NMR spectroscopy.

The dephosphorylation of phospho-amino acids with alkaline phosphatase (AlPase) from calf intestine or Escherichia coli and the phosphorylation of bovine serum albumin (BSA) with epidermal growth factor (EGF) receptor kinase from human A431 epidermoid carcinoma cells were investigated by 31P NMR spectroscopy. The initial rates of the dephosphorylation of phospho-tyrosine (P-Tyr) and phosphoserine (P-Ser) with AlPase were essentially the same in the one-substrate system. In the two-substrate system (P-Tyr plus P-Ser), however, the ratio of the initial rate for P-Tyr vs. P-Ser was 2.4 to 4.5 depending on the buffer and pH conditions employed. This substantiates for the first time the specificity of AlPases to P-Tyr over P-Ser at the free amino acid level. In the stationary phase of the overall process, the dephosphorylation of P-Ser became slow compared to that of P-Tyr in the one-substrate system. The decrease in the rate for P-Ser was further pronounced in the two-substrate system. For this remarkable effect, the rephosphorylation of serine was responsible, as demonstrated in the reaction mixture containing serine, Pi, and AlPase. BSA phosphorylated by EGF receptor kinase exhibited sharp 31P resonances around 0 ppm at neutral pH, far distant from the peak positions (4.9 ppm) of histone H1 phosphorylated by cAMP-dependent protein kinase. These NMR data are directed evidence that BSA was phosphorylated exclusively at the tyrosyl residues, whereas the phosphorylation of histone H1 was at the seryl residues.

Surface affinity chromatographic separation of blood cells. V. Retention behaviour of human peripheral blood cells on poly(propylene glycol) bonded agarose columns and its relationship to surface hydrophobicities of the gel beads and the cells.

Glycidyl ethers of poly(propylene glycol) (PPG) 200, 400 or 950 were oxirane-coupled with several kinds of agarose bead as the bonded phase of packing materials. An eluent was composed of 0.09 M phosphate-buffered 2% (w/w) dextran T40 solution at pH of respective isoelectric points of human blood cells. A linear relationship was found between the retention volumes of platelets, granulocytes and lymphocytes on the PPG-agarose columns and a measure of surface hydrophobicities of the cells, delta log K values, which were determined by using hydrophobic affinity partition. Furthermore, the retention volumes of granulocytes and lymphocytes increased according to the increase of delta log K of PPG-agarose beads. The retention of these two cells must be due to the hydrophobic interaction with the bonded PPG phase.