PubMed · 4098441
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1970-12-05. Mycobactins.. https://pubmed.ncbi.nlm.nih.gov/4098441/
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It has been proposed (S. Christen et al. Proc. Natl. Acad. Sci. USA 94, 3217-3222, 1997) that although alpha-tocopherol (alpha-TH) is an efficient antioxidant, the presence of gamma-tocopherol (gamma-TH) may be required to scavenge peroxynitrite-derived reactive nitrogen species. To investigate the reactions between alpha-TH, gamma-TH, and peroxynitrite, endogenous levels of both alpha-TH and gamma-TH were monitored when low-density lipoprotein was oxidized in the presence of the peroxynitrite generator 5-amino-3-(4-morpholinyl)-1, 2,3-oxadiazolium (SIN-1). SIN-1 oxidized alpha-TH while gamma-TH levels remained constant. The sparing of gamma-TH was also demonstrated when 1,2-dilauroyl-sn-glycero-3-phosphocholine liposomes containing alpha-TH and gamma-TH were incubated with either SIN-1 or peroxynitrite. Our data show that alpha-TH inhibits peroxynitrite-mediated gamma-TH nitration, i.e., 5-NO2-gamma-tocopherol formation. The rate constants for the reactions between both alpha-TH and gamma-TH with peroxynitrite suggest that the sparing of gamma-TH by alpha-TH does not occur by competitive scavenging, but may be due to the formation of a transient gamma-TH intermediate. Nitration of gamma-TH becomes significant only after alpha-TH levels have been depleted. We conclude alpha-TH alone is sufficient to remove any peroxynitrite-derived reactive nitrogen species, as the presence of alpha-TH attenuates nitration of both gamma-TH and tyrosine. The present results also indicate that a bolus addition of peroxynitrite or SIN-1 to liposomes containing gamma-TH forms 5-NO2-gamma-tocopherol in similar yields. This is in contrast to their reaction profile with tyrosine in aqueous solution. Under these conditions, SIN-1 does not form nitrotyrosine at detectable yields.
The homodimeric tetracycline repressor (TetR) regulates resistance to the antibiotic tetracycline at the transcriptional level. TetR binds in the absence of Tc to palindromic operator sequences utilizing two helix-turn-helix (HTH) motifs. If the tetracycline-Mg2+ complex [MgTc]+ enters two identical binding tunnels buried within the TetR homodimer, a conformational change takes place, and the induced [TetR/[MgTc]+]2 complex releases operator DNA. To demonstrate the contribution of Mg2+ to [MgTc]+ binding and TetR induction, the Mg2+ concentration in the induced TetR homodimer was progressively reduced by addition of EDTA, resulting in two X-ray crystal structures of Mg2+-free and half-occupied TetR(D). Tc remains bound to the [MgTc]+-binding sites, despite the complete or partial absence of Mg2+. Together with inducer-free TetR(D), the structures were refined to between 2.2 and 2.7 A resolution and compared with fully induced TetR(D) in complex with two [MgTc]+. Each inducer binding tunnel has three constituent parts, one hydrophobic and two hydrophilic ones. One of the hydrophilic contact areas binds Tc by hydrogen bonding; the hydrophobic region correctly positions Tc and partially closes the entrance to the binding tunnel; the second hydrophilic region coordinates Mg2+, transduces the induction signal, and completes the process of closing the tunnel entrance. Tc confers binding specificity to TetR while Mg2+ is primarily responsible for induction: After binding to the imidazole Nepsilon of His100, Mg2+ is octahedrally coordinated to the 1,3-ketoenolate group of Tc and to three water molecules. One of these waters forms a hydrogen bond to the hydroxyl group Ogamma of Thr103. The induced 2.5 A movement of Thr103 results in the partial unwinding of helix alpha6, associated with a lateral shift of helices alpha4 and alpha9. They simultaneously close the tunnel entrance and cause the DNA-binding domains to adopt a nonbinding conformation, leading to release of operator DNA and expression of the genes responsible for resistance.
There are several methods for locating the RNA site where a protein binds. One of the less common methods is directed cleavage of the RNA by an EDTA-Fe reagent tethered to the protein. The reaction of the EDTA-Fe(III) with ascorbate or hydrogen peroxide produces reactive oxygen species, such as hydroxyl radicals, localized within a 10-A radius of the iron center. The reactive oxygen species will attack the ribose or deoxyribose of nucleic acids as well as proximal polypeptide backbones. One EDTA-Fe reagent, (EDTA-2-aminoethyl)-2-pyridyl disulfide complexed to iron (EPD-Fe), has been tethered to several proteins through a disulfide linkage to engineered cysteine thiols and used to cleave DNA, proteins, and RNA. A second tethered EDTA-Fe reagent, 1-(p-bromoacetamidobenzyl)-EDTA-Fe, or BABE, has also been used to cleave RNA. Here we describe the issues involved in using these reagents with any RNA binding protein.