Heme ligation and conformational plasticity in the isolated c domain of cytochrome cd1 nitrite reductase

The heme ligation in the isolated c domain of Paracoccus pantotrophus cytochrome cd(1) nitrite reductase has been characterized in both oxidation states in solution by NMR spectroscopy. In the reduced form, the heme ligands are His69-Met106, and the tertiary structure around the c heme is similar to that found in reduced crystals of intact cytochrome cd1 nitrite reductase. In the oxidized state, however, the structure of the isolated c domain is different from the structure seen in oxidized crystals of intact cytochrome cd1, where the c heme ligands are His69-His17. An equilibrium mixture of heme ligands is present in isolated oxidized c domain. Two-dimensional exchange NMR spectroscopy shows that the dominant species has His69-Met106 ligation, similar to reduced c domains. This form is in equilibrium with a high-spin form in which Met106 has left the heme iron. Melting studies show that the midpoint of unfolding of the isolated c domain is 320.9 +/- 1.2 K in the oxidized and 357.7 +/- 0.6 K in the reduced form. The thermally denatured forms are high-spin in both oxidation states. The results reveal how redox changes modulate conformational plasticity around the c heme and show the first key steps in the mechanism that lead to ligand switching in the holoenzyme. This process is not solely a function of the properties of the c domain. The role of the d1 heme in guiding His17 to the c heme in the oxidized holoenzyme is discussed.     

2D:4D finger-length ratios in children and adults with gender identity disorder

Previous research suggests that prenatal testosterone affects the 2D:4D finger ratio in humans, and it has been speculated that prenatal testosterone also affects gender identity differentiation. If both things are true, then one would expect to find an association between the 2D:4D ratio and gender identity. We measured 2D:4D in two samples of patients with gender identity disorder (GID). In Study 1, we compared the 2D:4D ratios of 96 adult male and 51 female patients with GID to that of 90 heterosexual male and 112 heterosexual female controls. In Study 2, we compared the 2D:4D ratios of 67 boys and 34 girls with GID to that of 74 control boys and 72 control girls. In the sample of adults with GID, we classified their sexual orientation as either homosexual or non-homosexual (in relation to their birth sex) to examine whether or not there were any within-group differences as a function of sexual orientation. In the sample of adult men with GID (both homosexual and non-homosexual) and children with GID, we found no evidence of an altered 2D:4D ratio relative to same-sex controls. However, women with GID had a significantly more masculinized ratio compared to the control women. This last finding was consistent with the prediction that a variance in prenatal hormone exposure contributes to a departure from a sex-typical gender identity in women.     

The Saccharomyces cerevisiae Wss1 protein is only present in mother cells

The Saccharomyces cerevisiae WSS1 (Weak Suppressor of Smt3) gene has initially been identified as a multicopy suppressor of a mutation in SMT3 encoding the small ubiquitin-like modifier. Later, multiple functions related to DNA replication and repair have been found for WSS1. Here, we report the subcellular location of the Wss1 protein. Fluorescence microscopy of strains expressing a Wss1p-green fluorescent protein (GFP) fusion shows that the protein is present in a single sharp spot near the nuclear membrane, distinct from the spindle pole bodies and nucleolus. In dividing cells, the spot is exclusively present in the mother cell, suggesting a mother cell-specific function of WSS1.     

Copper(II) compounds of the planar-tridentate ligand 2,6-bis(pyrazol-3-yl)pyridine

The tridentate ligand 2,6-bis(pyrazol-3-yl)pyridine (dPzPy) renders coordination compounds with halide, nitrate and tetrafluoroborate salts of copper. The complexes, which have the form [Cu(dPzPy)X₂] with X=Br and Cl, [Cu(dPzPy)(NO₃)₂](H₂O), and [Cu(dPzPy)₂](BF₄)(SiF₆)_0.5(MeOH)₃ have been characterized by elemental analysis and by IR, EPR and ligand field spectroscopy. The single-crystal X-ray structure of [Cu(C₁₁H₉N₅)Br₂] shows the copper(II) ion to be coordinated by three N atoms of 2,6-bis(pyrazol-3-yl)pyridine and two bromides in a geometry exactly in between a trigonal-bipyramid and a square-pyramid. Each molecule lies on a crystallographic C₂-symmetry axis. They are coupled to one another by a two-dimensional network through NH to Br hydrogen bonds. The crystal structure of [Cu(C₁₁H₉N₅)Cl₂] is analogous to the bromide. The single-crystal X-ray structure of [Cu(dPzPy)₂](BF₄)(SiF₆)_0.5(MeOH)₃ shows the copper ion to be in a Jahn-Teller distorted octahedral N₆ environment of two mer-oriented tridentate ligands.     

The equilibrium unfolding of Azotobacter vinelandii apoflavodoxin II occurs via a relatively stable folding intermediate.

A flavodoxin from Azotobacter vinelandii is chosen as a model system to study the folding of alpha/beta doubly wound proteins. The guanidinium hydrochloride induced unfolding of apoflavodoxin is demonstrated to be reversible. Apoflavodoxin thus can fold in the absence of the FMN cofactor. The unfolding curves obtained for wild-type, C69A and C69S apoflavodoxin as monitored by circular dichroism and fluorescence spectroscopy do not coincide. Apoflavodoxin unfolding occurs therefore not via a simple two-state mechanism. The experimental data can be described by a three-state mechanism of apoflavodoxin equilibrium unfolding in which a relatively stable intermediate is involved. The intermediate species lacks the characteristic tertiary structure of native apoflavodoxin as deduced from fluorescence spectroscopy, but has significant secondary structure as inferred from circular dichroism spectroscopy. Both spectroscopic techniques show that thermally-induced unfolding of apoflavodoxin also proceeds through formation of a similar molten globule-like species. Thermal unfolding of apoflavodoxin is accompanied by anomalous circular dichroism characteristics: the negative ellipticity at 222 nM increases in the transition zone of unfolding. This effect is most likely attributable to changes in tertiary interactions of aromatic side chains upon protein unfolding. From the presented results and hydrogen/deuterium exchange data, a model for the equilibrium unfolding of apoflavodoxin is presented.     

Apparent local stability of the secondary structure of Azotobacter vinelandii holoflavodoxin II as probed by hydrogen exchange: implications for redox potential regulation and flavodoxin folding.

As a first step to determine the folding pathway of a protein with an alpha/beta doubly wound topology, the 1H, 13C, and 15N backbone chemical shifts of Azotobacter vinelandii holoflavodoxin II (179 residues) have been determined using multidimensional NMR spectroscopy. Its secondary structure is shown to contain a five-stranded parallel beta-sheet (beta2-beta1-beta3-beta4-beta5) and five alpha-helices. Exchange rates for the individual amide protons of holoflavodoxin were determined using the hydrogen exchange method. The amide protons of 65 residues distributed throughout the structure of holoflavodoxin exchange slowly at pH* 6.2 [kex < 10(-5) s(-1)] and can be used as probes in future folding studies. Measured exchange rates relate to apparent local free energies for transient opening. We propose that the amide protons in the core of holoflavodoxin only exchange by global unfolding of the apo state of the protein. The results obtained are discussed with respect to their implications for flavodoxin folding and for modulation of the flavin redox potential by the apoprotein. We do not find any evidence that A. vinelandii holoflavodoxin II is divided into two subdomains based on its amide proton exchange rates, as opposed to what is found for the structurally but not sequentially homologous alpha/beta doubly wound protein Che Y.     

An evaluation of the rate constants for the adsorption of the defective phage PBS Z to Bacillus subtilis

The adsorption of the defective phage PBS Z of Bacillus subtilis has previously been assumed to proceed in two steps, a reversible adsorption of extended phages followed by contraction of the adsorbed particles (Steensma, 1981a). This model, also used for other phages, explained the biphasic character of the adsorption curve, but a discrepancy was found between the calculated and observed concentrations of adsorbed, extended phages. Computer simulations indicated that this might be caused by inhomogeneity of the phage preparations with respect to their adsorption properties and that in that case other models would also fit the experimental data. Discrimination between the models was not possible on the basis of the available information on PBS Z and it was therefore concluded that the values reported previously for the rate constants (Steensma, 1981a) should be used with caution.     

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and analysis of the CEN1-ADE1-CDC15 region.

To continue the systematic examination of the physical and genetic organization of an entire Saccharomyces cerevisiae chromosome, the DNA from the CEN1-ADE1-CDC15 region from chromosome I was isolated and characterized. Starting with the previously cloned ADE1 gene (J. C. Crowley and D. B. Kaback, J. Bacteriol. 159:413-417, 1984), a series of recombinant lambda bacteriophages containing 82 kilobases of contiguous DNA from chromosome I were obtained by overlap hybridization. The cloned sequences were mapped with restriction endonucleases and oriented with respect to the genetic map by determining the physical positions of the CDC15 gene and the centromeric DNA (CEN1). The CDC15 gene was located by isolating plasmids from a YCp50 S. cerevisiae genomic library that complemented the cdc15-1 mutation. S. cerevisiae sequences from these plasmids were found to be represented among those already obtained by overlap hybridization. The cdc15-1-complementing plasmids all shared only one intact transcribed region that was shown to contain the bona fide CDC15 gene by in vitro gene disruption and one-step replacement to delete the chromosomal copy of this gene. This deletion produced a recessive lethal phenotype that was also recessive to cdc15-1. CEN1 was located by finding a sequence from the appropriate part of the cloned region that stabilized the inheritance of autonomously replicating S. cerevisiae plasmid vectors. Finally, RNA blot hybridization and electron microscopy of R-loop-containing DNA were used to map transcribed regions in the 23 kilobases of DNA that went from CEN1 to CDC15. In addition to the transcribed regions corresponding to the ADE1 and ADC15 genes, this DNA contained five regions that gave rise to polyadenylated RNA, at least two regions complementary to 4S RNA species, and a Ty1 transposable element. Notably, a higher than average proportion of the DNA examined was transcribed into RNA.     

The quaternary structure of the sheaths of defective phages similar to PBS X.

The contractile sheaths of five defective, PBS X-like bacteriophages from Bacillus subtilis and B. licheniformis were investigated by electron microscopy, dodecylsulphate gel electrophoresis and immunodiffusion. Electron microscope images of the extended and contracted sheaths were of similar appearance, although their lengths were different. The surface lattices of both the extended and the contracted sheaths were determined by optical diffraction. This showed that the quaternary structure of the sheaths of all five defective phages originated from identical surface lattices, which could be approximately expressed by the selection rules L = -2n' + 3m and L = 9N' + 17M for the extended and contracted sheaths respectively, in which 6n' = n with n = 0 or an integer multiple of 6. These results indicated that the packing of the protein subunits in these sheaths differed from those of other bacteriophages, for example T4 and millimicron [Amos and Klug, J. Mol. Biol. 99, 51--73 (1975); Admiraal and Mellema, J. Ultrastruct. Res. 56, 48--64 (1976)]. The molecular weight of the main sheath protein of the defective phages, as determined by dodecylsulphate gel electrophoresis, was approximately 50000. This value differed from that for T4, but was similar to that of millimicron [Admiraal and Mellema, J. Ultrastruct. Res. 56, 48--64 (1976); King and Laemmli, J. Mol. Biol, 75, 315--337 (1973)]. The results of immunodiffusion experiments, however, pointed to a chemical difference between the sheath proteins of the defective phages and millimicron, in addition to T4.     

The occurrence and taxonomic value of PBS X-like defective phages in the genus Bacillus

72 strains of 24 Bacillus species were induced with mitomycin C. The lysates were examined for the presence of defective phages resembling PBS X in morphology. All strains tested of B. amyloliquefaciens. B, licheniformis, B. pumilus and B. subtilis contained such phages. Five morphological types of defective, PBS X-like phage could be distinguished, differing in their tail lengths and in the number of cross-striations on the tail. The quaternary structure of the tail, the molecular weight of the main tail protein and the antigenic properties of the phages were identical. The killing ranges of the defective phages have been determined and their possible use in taxonomy discussed.