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.

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.

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 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.

Multiple conformations coexist and interchange in natural B DNA fibers.

31P nuclear magnetic resonance (n.m.r.) of highly oriented NaDNA and LiDNA fibers was measured as a function of relative humidity over the range from 66% to 98%. The humidity dependence of the spectral patterns of NaDNA fibers shows that the A form has a single conformation while the B form has multiple conformations, and that interconversion between the A and B conformers in the transition region is slow compared to the n.m.r. time-scale (approximately 10(-5) s). Two major conformations of the B form of LiDNA are found to be stable at low relative humidity and they rapidly interchange at high relative humidities. The spectral patterns of immobilized LiDNA are compatible with the single-crystal structure of double-stranded oligo nucleic acids.

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.

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.

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.

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.