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Site-directed Mutants (site-directed + mutant)
Selected AbstractsFerritin ferroxidase activity: A potent inhibitor of osteogenesisJOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2010Abolfazl Zarjou Abstract Hemochromatosis is a known cause of osteoporosis, and iron overload has deleterious effects on bone. Although iron overload and its association with osteoporosis has long been recognized, the pathogenesis and exact role of iron have been undefined. Bone is an active tissue with constant remodeling capacity. Osteoblast (OB) development and maturation are under the influence of core binding factor ,-1 (CBF-,1), which induces expression of OB-specific genes, including alkaline phosphatase, an important enzyme in early osteogenesis, and osteocalcin, a noncollagenous protein deposited within the osteoid. This study investigates the mechanism by which iron inhibits human OB activity, which in vivo may lead to decreased mineralization, osteopenia, and osteoporosis. We demonstrate that iron-provoked inhibition of OB activity is mediated by ferritin and its ferroxidase activity. We confirm this notion by using purified ferritin H-chain and ceruloplasmin, both known to possess ferroxidase activity that inhibited calcification, whereas a site-directed mutant of ferritin H-chain lacking ferroxidase activity failed to provide any inhibition. Furthermore, we are reporting that such suppression is not restricted to inhibition of calcification, but OB-specific genes such as alkaline phosphatase, osteocalcin, and CBF-,1 are all downregulated by ferritin in a dose-responsive manner. This study corroborates that iron decreases mineralization and demonstrates that this suppression is provided by iron-induced upregulation of ferritin. In addition, we conclude that inhibition of OB activity, mineralization, and specific gene expression is attributed to the ferroxidase activity of ferritin. © 2010 American Society for Bone and Mineral Research [source] Transient Vibronic Structure in Ultrafast Fluorescence Spectra of Photoactive Yellow Protein,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008Ryosuke Nakamura The ultrafast photo-induced dynamics of wild-type photoactive yellow protein and its site-directed mutant of E46Q in aqueous solution was studied at room temperature by femtosecond fluorescence spectroscopy using the optical Kerr-gate method. The vibronic structure appears, depending on the excitation photon energy, in the time-resolved fluorescence spectra just after photoexcitation, which winds with time and disappears on a time scale of sub-picoseconds. This result indicates that the wavepacket is localized in the electronic excited state followed by dumped oscillations and broadening, and also that the initial condition of the wavepacket prepared depending on the excitation photon energy affects much the following ultrafast dynamics in the electronic excited state. [source] ATP-dependent modulation and autophosphorylation of rapeseed 2-Cys peroxiredoxinFEBS JOURNAL, Issue 7 2008Martin Aran 2-Cys peroxiredoxins (2-Cys Prx) are ubiquitous thiol-containing peroxidases that have been implicated in antioxidant defense and signal transduction. Although their biochemical features have been extensively studied, little is known about the mechanisms that link the redox activity and non-redox processes. Here we report that the concerted action of a nucleoside triphosphate and Mg2+ on rapeseed 2-Cys Prx reversibly impairs the peroxidase activity and promotes the formation of high molecular mass species. Using protein intrinsic fluorescence in the analysis of site-directed mutants, we demonstrate that ATP quenches the emission intensity of Trp179, a residue close to the conserved Cys175. More importantly, we found that ATP facilitates the autophosphorylation of 2-Cys Prx when the protein is successively reduced with thiol-bearing compounds and oxidized with hydroperoxides or quinones. MS analyses reveal that 2-Cys Prx incorporates the phosphoryl group into the Cys175 residue yielding the sulfinic-phosphoryl [Prx-(Cys175)-SO2PO32,] and the sulfonic-phosphoryl [Prx-(Cys175)-SO3PO32,] anhydrides. Hence, the functional coupling between ATP and 2-Cys Prx gives novel insights into not only the removal of reactive oxygen species, but also mechanisms that link the energy status of the cell and the oxidation of cysteine residues. [source] Assignment of the [4Fe-4S] clusters of Ech hydrogenase from Methanosarcina barkeri to individual subunits via the characterization of site-directed mutantsFEBS JOURNAL, Issue 18 2005Lucia Forzi Ech hydrogenase from Methanosarcina barkeri is a member of a distinct group of membrane-bound [NiFe] hydrogenases with sequence similarity to energy-conserving NADH:quinone oxidoreductase (complex I). The sequence of the enzyme predicts the binding of three [4Fe-4S] clusters, one by subunit EchC and two by subunit EchF. Previous studies had shown that two of these clusters could be fully reduced under 105 Pa of H2 at pH 7 giving rise to two distinct S½ electron paramagnetic resonance (EPR) signals, designated as the g = 1.89 and the g = 1.92 signal. Redox titrations at different pH values demonstrated that these two clusters had a pH-dependent midpoint potential indicating a function in ion pumping. To assign these signals to the subunits of the enzyme a set of M. barkeri mutants was generated in which seven of eight conserved cysteine residues in EchF were individually replaced by serine. EPR spectra recorded from the isolated mutant enzymes revealed a strong reduction or complete loss of the g = 1.92 signal whereas the g = 1.89 signal was still detectable as the major EPR signal in five mutant enzymes. It is concluded that the cluster giving rise to the g = 1.89 signal is the proximal cluster located in EchC and that the g = 1.92 signal results from one of the clusters of subunit EchF. The pH-dependence of these two [4Fe-4S] clusters suggests that they simultaneously mediate electron and proton transfer and thus could be an essential part of the proton-translocating machinery. [source] The function of D1-H332 in Photosystem II electron transport studied by thermoluminescence and chlorophyll fluorescence in site-directed mutants of Synechocystis 6803FEBS JOURNAL, Issue 17 2004Yagut Allahverdiyeva The His332 residue of the D1 protein has been identified as the likely ligand of the catalytic Mn ions in the water oxidizing complex (Ferreira, K.N., Iverson, T.M., Maghlaoui, K., Barber, J. & Iwata, S. (2004) Science 303, 1831,1838). However, its function has not been fully clarified. Here we used thermoluminescence and flash-induced chlorophyll fluorescence measurements to characterize the effect of the D1-H333E, D1-H332D and D1-H332S mutations on the electron transport of Photosystem II in intact cells of the cyanobacterium Synechocystis 6803. Although the mutants are not photoautotrophic they all show flash-induced thermoluminescence and chlorophyll fluorescence, which originate from the S2QA, and S2QB, recombinations demonstrating that charge stabilization takes place in the water oxidizing complex. However, the conversion of S2 to higher S states is inhibited and the energetic stability of the S2QA, charge pair is increased by 75, 50 and 7 mV in the D1-H332D, D1-H332E and D1-H332S mutants, respectively. This is most probably caused by a decrease of Em(S2/S1). Concomitantly, the rate of electron donation from Mn to Tyr-Z, during the S1 to S2 transition is slowed down, relative to the wild type, 350- and 60-fold in the D1-H332E and D1-H332D mutants, respectively, but remains essentially unaffected in D1-H332S. A further effect of the D1-H332E and D1-H332D mutations is the retardation of the QA to QB electron transfer step as an indirect consequence of the donor side modification. Our data show that although the His residue in the D1-332 position can be substituted by other metal binding residues for binding photo-oxidisable Mn it is required for controlling the functional redox energetics of the Mn cluster. [source] Spondylarthritis-associated and non,spondylarthritis-associated B27 subtypes differ in their dependence upon tapasin for surface expression and their incorporation into the peptide loading complexARTHRITIS & RHEUMATISM, Issue 1 2006Jane C. Goodall Objective B27 subtypes associated with susceptibility to ankylosing spondylitis (AS), and those reported not to be associated with AS, are found to differ in the amino acids that are known in other HLA class I molecules to alter the requirements for tapasin and incorporation into the peptide loading complex. The purpose of this study was to examine the behavior of B*2704 and B*2705 in comparison with B*2706 and B*2709 during early events in HLA class I antigen expression, and determine if their behavior correlates with disease association. Methods Cell lines with nonfunctional tapasin were transiently transfected with different B27 subtypes and their site-directed mutants, and surface expression analyzed by flow cytometry. The association with the peptide loading complex was determined by immunoprecipitation of heterodimeric transporter-associated peptide and analysis of coprecipitated B27. Results Amino acids at positions 114, 116, and 152 in the different B27 subtypes were shown to perform key roles in defining a requirement for interaction with tapasin. Not all disease-associated alleles were expressed optimally in the absence of tapasin; furthermore, dependence on tapasin for cell surface expression did not correlate with disease association. Although B*2706, which is not associated with disease, exhibited a number of properties different from those of the disease-associated subtypes, these properties were not displayed by the non,disease-associated allele B*2709. Conclusion These results indicate that the ability to exhibit optimal cell surface expression in the absence of tapasin is not a prerequisite for susceptibility to AS. [source] | ||||||||||