44 research outputs found
A Distance Ruler for RNA Using EPR and Site-Directed Spin Labeling
AbstractAs a basic model study for measuring distances in RNA molecules using continuous wave (CW) EPR spectroscopy, site-directed spin-labeled 10-mer RNA duplexes and HIV-1 TAR RNA motifs with various interspin distances were examined. The spin labels were attached to the 2â˛-NH2 positions of appropriately placed uridines in the duplexes, and interspin distances were measured from both molecular dynamics simulations (MD) and Fourier deconvolution methods (FD) [13]. The 10-mer duplexes have interspin distances ranging from 10 Ă
to 30 Ă
based on MD; however, dipolar line broadening of the CW EPR spectrum is only observed for the RNAs for predicted interspin distances of 10â21 Ă
and not for distances over 25 Ă
. The conformational changes in TAR (transactivating responsive region) RNA in the presence and in the absence of different divalent metal ions were monitored by measuring distances between two nucleotides in the bulge region. The predicted interspin distances obtained from the FD method and those from MD calculations match well for both the model RNA duplexes and the structural changes predicted for TAR RNA. These results demonstrate that distance measurement using EPR spectroscopy is a potentially powerful method to help predict the structures of RNA molecules
Protonation of the Binuclear Metal Center within the Active Site of Phosphotriesterase â
ABSTRACT: Phosphotriesterase (PTE) is a binuclear metalloenzyme that catalyzes the hydrolysis of organophosphates, including pesticides and chemical warfare agents, at rates approaching the diffusion controlled limit. The catalytic mechanism of this enzyme features a bridging solvent molecule that is proposed to initiate nucleophilic attack at the phosphorus center of the substrate. X-band EPR spectroscopy is utilized to investigate the active site of Mn/Mn-substituted PTE. Simulation of the dominant EPR spectrum from the coupled binuclear center of Mn/Mn-PTE requires slightly rhombic zero-field splitting parameters. Assuming that the signal arises from the S ) 2 manifold, an exchange coupling constant of J ) -2.7 ( 0.2 cm -1 (H ex ) -2JS 1 âS 2 ) is calculated. A kinetic pK a of 7.1 ( 0.1 associated with loss in activity at low pH indicates that a protonation event is responsible for inhibition of catalysis. Analysis of changes in the EPR spectrum as a function of pH provides a pK a of 7.3 ( 0.1 that is assigned as the protonation of the hydroxyl bridge. From the comparison of kinetic and spectral pK a values, it is concluded that the loss of catalytic activity at acidic pH results from the protonation of the hydroxide that bridges the binuclear metal center. Phosphotriesterase (PTE) 1 catalyzes the hydrolysis of a wide range of organophosphate esters, including agricultural pesticides and chemical warfare agents (1-3). The enzyme has been isolated from soil bacteria, but the natural substrate for PTE is not known. PTE is a member of the amidohydrolase superfamily, which also includes urease, dihydroorotase, and approximately 30 other enzymes of known specificity (4). The high-resolution X-ray crystal structure of Zn/Zn-PTE reveals that it is a homodimeric protein containing an active site with two divalent metal ions embedded within a ( /R) 8 -barrel motif (5). The R-metal ion is ligated by His-55, His-57, and Asp-301 while the -metal ion is coordinated to His-201 and His-230 as illustrated i
Characterization of a native hammerhead ribozyme derived from schistosomes
A recent re-examination of the role of the helices surrounding the conserved core of the hammerhead ribozyme has identified putative loopâloop interactions between stems I and II in native hammerhead sequences. These extended hammerhead sequences are more active at low concentrations of divalent cations than are minimal hammerheads. The loopâloop interactions are proposed to stabilize a more active conformation of the conserved core. Here, a kinetic and thermodynamic characterization of an extended hammerhead sequence derived from Schistosoma mansoni is performed. Biphasic kinetics are observed, suggesting the presence of at least two conformers, one cleaving with a fast rate and the other with a slow rate. Replacing loop II with a poly(U) sequence designed to eliminate the interaction between the two loops results in greatly diminished activity, suggesting that the loopâloop interactions do aid in forming a more active conformation. Previous studies with minimal hammerheads have shown deleterious effects of R(p)-phosphorothioate substitutions at the cleavage site and 5Ⲡto A9, both of which could be rescued with Cd(2+). Here, phosphorothioate modifications at the cleavage site and 5Ⲡto A9 were made in the schistosome-derived sequence. In Mg(2+), both phosphorothioate substitutions decreased the overall fraction cleaved without significantly affecting the observed rate of cleavage. The addition of Cd(2+) rescued cleavage in both cases, suggesting that these are still putative metal binding sites in this native sequence
Platinum Binds Proteins in the Endoplasmic Reticulum of <i>S. cerevisiae</i> and Induces Endoplasmic Reticulum Stress
PtÂ(II)-based
anticancer drugs are widely used in the treatment
of a variety of cancers, but their clinical efficacy is hindered by
undesirable side effects and resistance. While much research has focused
on PtÂ(II) drug interactions with DNA, there is increasing interest
in proteins as alternative targets and contributors to cytotoxic and
resistance mechanisms. Here, we describe a chemical proteomic method
for isolation and identification of cellular protein targets of platinum
compounds using PtÂ(II) reagents that have been modified for participation
in the 1,3 dipolar cycloaddition âclickâ reaction. Using
this method to visualize and enrich for targets, we identified 152
proteins in PtÂ(II)-treated <i>Saccharomyces cerevisiae</i>. Of interest was the identification of multiple proteins involved
in the endoplasmic reticulum (ER) stress response, which has been
proposed to be an important cytoplasmic mediator of apoptosis in response
to cisplatin treatment. Consistent with possible direct targeting
of this pathway, the ER stress response was confirmed to be induced
in PtÂ(II)-treated yeast along with <i>in vitro</i> PtÂ(II)-inhibition
of one of the identified proteins, protein disulfide isomerase
Site-Specific Platinum(II) Cross-Linking in a Ribozyme Active Site
The function of RNA depends on its ability to adopt complex and dynamic structures, and the incorporation of site-specific cross-linking probes is a powerful method for providing distance constraints that are valuable in RNA structural biology. Here we describe a new RNAâRNA cross-linking strategy based on Pt(II) targeting of specific phosphorothioate substitutions. In this strategy <i>cis</i>-diammine Pt(II) complexes are kinetically recruited and anchored to a phosphorothioate substitution embedded within a structured RNA. Substitution of the remaining exchangeable Pt(II) ligand with a nucleophile supplied by a nearby RNA nucleobase results in metal-mediated cross-links that are stable during isolation. This type of cross-linking strategy was explored within the catalytic core of the Hammerhead ribozyme (HHRz). When a phosphorothioate substitution is installed at the scissile bond normally cleaved by the HHRz, Pt(II) cross-linking takes place to nucleotides G8 and G10 in the ribozyme active site. Both of these positions are predicted to be within âź8 Ă
of a phosphorothioate-bound Pt(II) metal center. Cross-linking depends on Mg<sup>2+</sup> ion concentration, reaching yields as high as 30%, with rates that indicate cation competition within the RNA three-helix junction. Cross-linking efficiency depends on accurate formation of the HHRz tertiary structure, and cross-links are not observed for RNA helices. Combined, these results show promise for using kinetically inert Pt(II) complexes as new site-specific cross-linking tools for exploring RNA structure and dynamics
Platinum Binds Proteins in the Endoplasmic Reticulum of <i>S. cerevisiae</i> and Induces Endoplasmic Reticulum Stress
PtÂ(II)-based
anticancer drugs are widely used in the treatment
of a variety of cancers, but their clinical efficacy is hindered by
undesirable side effects and resistance. While much research has focused
on PtÂ(II) drug interactions with DNA, there is increasing interest
in proteins as alternative targets and contributors to cytotoxic and
resistance mechanisms. Here, we describe a chemical proteomic method
for isolation and identification of cellular protein targets of platinum
compounds using PtÂ(II) reagents that have been modified for participation
in the 1,3 dipolar cycloaddition âclickâ reaction. Using
this method to visualize and enrich for targets, we identified 152
proteins in PtÂ(II)-treated <i>Saccharomyces cerevisiae</i>. Of interest was the identification of multiple proteins involved
in the endoplasmic reticulum (ER) stress response, which has been
proposed to be an important cytoplasmic mediator of apoptosis in response
to cisplatin treatment. Consistent with possible direct targeting
of this pathway, the ER stress response was confirmed to be induced
in PtÂ(II)-treated yeast along with <i>in vitro</i> PtÂ(II)-inhibition
of one of the identified proteins, protein disulfide isomerase