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The proximity of Ca to the Mn cluster of the photosynthetic water-oxidation complex is demonstrated by X-ray absorption spectroscopy. We have collected EXAFS data at the Ca K-edge using active PS II membrane samples that contain approximately 2 Ca per 4 Mn. These samples are much less perturbed than previously investigated Sr-substituted samples, which were prepared after Ca depletion. The new Ca EXAFS clearly shows backscattering from Mn at 3.4 Å, a distance that agrees with that surmised from previously recorded Mn EXAFS. This result is also consistent with earlier related experiments at the Sr K-edge, using samples that contained functional Sr, that show Mn is ~3.5 Å distant from Sr. The totality of the evidence clearly advances the notion that the catalytic center of oxygen evolution is a Mn-Ca heteronuclear cluster. [ABSTRACT FROM AUTHOR]

Here we present a split-enzyme sensor approach for the sequence-specific detection of metal-based drug adducts of DNA. Split β-lactamase reporters were constructed using domain A of the High Mobility Group Box 1 protein (HMGB1a) in conjunction with zinc finger DNA-binding domains. As a proof of concept, the sensors were characterized with the well-known drug cisplatin, which forms 1,2-intrastrand crosslinks with DNA that are recognized by HMGB1a. After promising results with cisplatin, five ruthenium-based drugs were studied, four of which produced significant signal over background. These results highlight the utility of our approach for rapid screening of novel metal-based chemotherapeutic drug candidates and provide evidence that HMGB1a likely binds to DNA adducts formed by NAMI-A (imidazolium trans -tetrachlorodimethylsulfoxideimidazoleruthenate(III)), KP1019 (indazolium trans -tetrachlorodiindazoleruthenate(III)), KP418 (imidazolium trans -tetrachlorodiimidazoleruthenate(III)), and RAPTA-C (dichloro(η6- p -cymene)(1,3,5-triaza-7-phosphaadamantane)ruthenium(II)). These results thus imply a potential biologically relevant mode of action for the ruthenium-based drugs investigated herein. Split-enzyme sensors with the capacity to detect metal-based adducts of DNA have been developed. The sensors use a split β-lactamase reporter coupled to High Mobility Group Box 1 protein domain A (HMGB1a) partnered with a zinc finger DNA binding domain. Several dsDNA adducts of cisplatin and ruthenium-based drugs were investigated. [Display omitted] • Domain A of the High Mobility Group Box 1 protein (HMGB1a) binds to cisplatin-modified DNA. • Split protein sensors using HMGB1a can detect modification of DNA by cisplatin. • Sensors for cisplatin-modified DNA are modular and can detect various dsDNA targets. • Sensors that detect cisplatin-modified DNA also respond to DNA reacted with Ru drugs. • HMGB1a likely binds to DNA modified by some ruthenium-based drugs. [ABSTRACT FROM AUTHOR]

The article focuses on the changes in the conformation of tRNAPhe by using an integrated biochemistry laboratory course. Students use three experimental techniques including gel electrophoresis, UV-vis spectrophotometry, and fluorescence spectroscopy. The class examines the changes in the structure of a ligand that affect its ability to bind and change the tRNAPhe structure. Discussions concerning the projects probing tRNAPhe structure using various assay methods are also presented.

The ruthenium-based anticancer agent NAMI-A (ImH[ trans -RuCl 4 (dmso)(Im)], where Im = imidazole) has been shown to interact with RNA in vivo and in vitro. We hypothesized that the similarly structured drug KP1019 (IndH[ trans -RuCl 4 (Ind) 2 ], where Ind = indazole) binds to RNA as well. Fluorescence spectroscopy was employed to assay the interactions between either NAMI-A or KP1019 and tRNA Phe through an intrinsic fluorophore wybutosine (Y) base and by extrinsic displacement of the intercalating agent ethidium bromide. In both the intrinsic Y-base and extrinsic ethidium bromide studies, KP1019 exhibited tighter binding to phenylalanine-specific tRNA (tRNA Phe ) than NAMI-A. In the ethidium bromide study, reducing both drugs from Ru III to Ru II resulted in a significant decrease in binding. Our findings suggest that the relatively large heteroaromatic indazole ligands of KP1019 intercalate in the π-stacks of tRNA Phe within structurally complex binding pockets. In addition, NAMI-A appears to be sensitive to destabilizing electrostatic interactions with the negative phosphate backbone of tRNA Phe . Interactions with additional tRNA molecules and other types of RNA require further evaluation to determine the role of RNA in the mechanisms of action for KP1019 and to better understand how Ru drugs fundamentally interact with biomolecules that are more structurally sophisticated than short DNA oligonucleotides. To the best of our knowledge, this is the first study to report KP1019 binding interactions with RNA. [ABSTRACT FROM AUTHOR]

This article presents a study regarding the fluorescence spectroscopy of tRNAPhe Y base in the presence of magnesium2+ and small molecule ligands. It discusses that tRNAPhe contains a unique Y base with three planar aromatic rings and serves as a fluorophore. It serves as an intrinsic probe to study the conformational changes of tRNAPhe in the presence of magnesium ions and small molecule ligands because Y base fluorescence is exquisitely sensitive to the local environment.

This article presents a study regarding the metal-catalyzed cleavage of tRNAPhe. It discusses that Lead(II)-catalyzed hydrolysis is an effective assay of tRNAPhe tertiary structure because it catalyzes the cleavage of native, folded tRNAPhe at a single site in the D loop resulting in a large fragment and a small fragment. In addition, ligand binding alters the conformation of tRNAPhe inhibiting lead-catalyzed cleavage and metals other than lead may also cleave tRNA.

This article presents a study regarding the UV thermal melting curves of tRNAPhe in the presence of ligands. It discusses that native secondary and tertiary structures of tRNAPhe are only stable below about 50-70 degrees celcius because of the positive entropy of unfolding. A laboratory project was designed to investigate the influence of equilibrium ligand binding on the conformational stability of tRNAPhe via UV melting curves. The melting temperature is derived by calculating the derivatives of the absorbance versus temperature curves using the Cary program.

[ImH][ trans-RuCl(DMSO)(Im)] (where DMSO is dimethyl sulfoxide and Im is imidazole) (NAMI-A) is an antimetastatic prodrug currently in phase II clinical trials. The mechanisms of action of this and related Ru-based anticancer agents are not well understood, but several cellular targets have been suggested. Although Ru has been observed to bind to DNA following in vitro NAMI-A exposure, little is known about Ru-DNA interactions in vivo and even less is known about how this or related metallodrugs might influence cellular RNA. In this study, Ru accumulation in cellular RNA was measured following treatment of Saccharomyces cerevisiae with NAMI-A. Drug-dependent growth and cell viability indicate relatively high tolerance, with approximately 40% cell death occurring at 6 h for 450 μM NAMI-A . Significant dose-dependent accumulation of Ru in cellular RNA was observed by inductively coupled plasma mass spectrometry measurements on RNA extracted from yeast treated with NAMI-A. In vitro, binding of Ru species to drug-treated model DNA and RNA oligonucleotides at pH 6.0 and 7.4 was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in the presence and absence of the reductant ascorbate. The extent of Ru-nucleotide interactions increases slightly with lower pH and significantly in the presence of ascorbate, with differences in observed species distribution. Taken together, these studies demonstrate the accumulation of aquated and reduced derivatives of NAMI-A on RNA in vitro and in cellulo, and enhanced binding with nucleic acid targets in a tumorlike acidic, reducing environment. To our knowledge, this is also the first study to characterize NAMI-A treatment of S. cerevisiae, a genetically tractable model organism. [ABSTRACT FROM AUTHOR]