Evaluation of a Highly Efficient Multidrug Biochip Array Technology for a Simultaneous and High-Throughput Urine Drug Screening in Clinical and Toxicological Settings.

BACKGROUND: A high-throughput and highly efficient analytical platform for urine drug screening is critical in both clinical and forensic settings. Mass spectrometry (MS) has better sensitivity and specificity than conventional immunoassays (IA); however, not all laboratories have the necessary resources and workforce to operate MS. The goal of this study was to evaluate a multidrug biochip with 20 discrete testing regions (DTRs) for high-throughput urine drug screening (UDS). METHODS: The Randox DOA Ultra Urine (DOAULT URN) biochip employs chemiluminescent IA to detect various analytes, including stimulants, hallucinogens, sedatives, narcotics, and dextromethorphan. The verification included the evaluation of the limits of detection (LOD), stability of calibrators and controls, cross-reactivity, carryover, interference, and overall performance. RESULTS: LODs < quality control low for each DTR. The reconstituted calibrators were stable for up to 2 weeks at -20°C. Controls were stable for 4-6 hours at 22-25°C, with <20% within-day and ≤23% between-day imprecision. The accuracy of the controls (%bias) was within ±20% of the target concentration, except for dextromethorphan at -23.8%. No interference was observed with common over-the-counter medications. No carryover was detected in the high-concentration samples. Satisfactory cross-reactivity (≥50%) with known analytes produced presumptive positive results, with readings higher than the proposed decision points. The overall biochip performance of 165 confirmed samples showed 98.0% sensitivity, 96.9% specificity, and 97.5% efficiency. CONCLUSIONS: The DOAULT URN biochip is a multidrug analyte IA capable of detecting dozens of parent drugs and their metabolites in urine. It offers clinical and forensic laboratories an alternative UDS tool with LODs comparable to those of MS.

A metal-ligand-mediated intersubunit allosteric switch in related SmtB/ArsR zinc sensor proteins.

The origin of metal ion selectivity by members of the SmtB/ArsR family of bacterial metal-sensing transcriptional repressors and the mechanism of negative allosteric regulation of DNA binding is poorly understood. Here, we report that two homologous zinc sensors, Staphylococcus aureus CzrA and cyanobacterial SmtB, are "winged" helix homodimeric DNA-binding proteins that bind Zn(II) to a pair of tetrahedral, interhelical binding sites, with two ligands derived from the alpha5 helix of one subunit, Asp84 O(delta1) (Asp104 in SmtB), His86 N(delta1) (His106), and two derived from the alpha5 helix of the other, His97' N(delta1) (His117') and His100' N(epsilon2) (Glu120'). Formation of the metal chelate drives a quaternary structural switch mediated by an intersubunit hydrogen-binding network that originates with the non-liganding N(epsilon2) face of His97 in CzrA (His117 in SmtB) that stabilizes a low-affinity, DNA-binding conformation. The structure of the Zn(1) SmtB homodimer shows that both metal-binding sites of the dimer must be occupied for the quaternary structural switch to occur. Thus, a critical zinc-ligating histidine residue obligatorily couples formation of the metal-sensing coordination chelate to changes in the conformation and dynamics of the putative DNA-binding helices.

Assembly of Ebola virus matrix protein VP40 is regulated by latch-like properties of N and C terminal tails.

The matrix protein VP40 coordinates numerous functions in the viral life cycle of the Ebola virus. These range from the regulation of viral transcription to morphogenesis, packaging and budding of mature virions. Similar to the matrix proteins of other nonsegmented, negative-strand RNA viruses, VP40 proceeds through intermediate states of assembly (e.g. octamers) but it remains unclear how these intermediates are coordinated with the various stages of the life cycle. In this study, we investigate the molecular basis of synchronization as governed by VP40. Hydrogen/deuterium exchange mass spectrometry was used to follow induced structural and conformational changes in VP40. Together with computational modeling, we demonstrate that both extreme N and C terminal tail regions stabilize the monomeric state through a direct association. The tails appear to function as a latch, released upon a specific molecular trigger such as RNA ligation. We propose that triggered release of the tails permits the coordination of late-stage events in the viral life cycle, at the inner membrane of the host cell. Specifically, N-tail release exposes the L-domain motifs PTAP/PPEY to the transport and budding complexes, whereas triggered C-tail release could improve association with the site of budding.

Structural characterization of distinct alpha3N and alpha5 metal sites in the cyanobacterial zinc sensor SmtB.

SmtB is required for Synechococcus to effect a response to toxic concentrations of Zn(II) and other heavy metals. Direct binding of inducing metal ions to SmtB transcriptionally derepresses the expression of SmtA, a prokaryotic class II metallothionein. Homodimeric SmtB binds one Zn(II) or Co(II) per monomer in a cysteine thiolate-containing site in a tetrahedral coordination geometry [VanZile, M. L., et al. (2000) Biochemistry 39, 11818-11829]. In this report, characterization of a set of cysteine substitution mutants of SmtB reveals that SmtB homodimer binds Zn(II) or Co(II) in one of two mutually exclusive metal binding sites, termed alpha3N and alpha5, with very high equilibrium affinities. Both sites are characterized by similar affinities for Co(II) (K(Co) approximately equal to 2-5 x 10(9) M(-1)), while the Zn(II) affinities are at least 20-fold different (K(Zn)(alpha)(3N) > or = 10(13) M(-1); K(Zn)(alpha)(5) approximately equal to 5 x 10(11) M(-1)). Co(II) bound exclusively at the alpha5 sites is capable of rapid equilibration between the alpha3N and alpha5 sites upon reduction of the mixed disulfides in S-methylated SmtB. These results suggest that the alpha3N or alpha5 metal sites might play distinct roles in this Zn(II)-sensing protein, systematically investigated in the following paper [VanZile, M. L., Chen, X., and Giedroc, D. P. (2002) Biochemistry 41, 9776-9786]. Since both the alpha3N and alpha5 sites are present in many members of the SmtB/ArsR family of metal sensor proteins, the presence of these two metal binding sites may explain some of the functional diversity in metal responses across this family of proteins.

Allosteric negative regulation of smt O/P binding of the zinc sensor, SmtB, by metal ions: a coupled equilibrium analysis.

The Synechococcus PCC 7942 smt operon is responsible for cellular resistance to excess zinc and consists of two divergently transcribed genes, smtB and smtA. SmtB is the Zn(II)-sensing metal-regulated repressor of the system and binds to a 12-2-12 imperfect inverted repeat in the smtA O/P region. Using fluorescence anisotropy to monitor SmtB-smt O/P multiple equilibria, we show that four SmtB homodimers bind to a 40 bp oligonucleotide containing a single 12-2-12 inverted repeat. The binding affinities of the first two dimers are very tight (K(int) = 2.9 x 10(9) M(-1)) with the affinities of the third and fourth dimers lower by approximately 10- and approximately 30-fold, respectively. A single monomer equivalent of Zn(II), Cd(II), or Co(II) promotes disassembly of the oligomeric complex to a mixture of (P(2)).D and (P(2))(2).D SmtB dimer-DNA complexes with the intrinsic affinity of all SmtB homodimers for DNA greatly reduced by approximately 500-2000-fold. Substitution or derivatization of cysteines which comprise the alpha3N metal binding site (Cys14 and Cys61) [VanZile, M. L., et al. (2002) Biochemistry 41, 9765-9775] has no effect on allosteric negative regulation by Zn(II); in contrast, H106Q SmtB, harboring a single zinc-liganding substitution in the alpha5 metal binding site, is refractory to zinc-induced disassembly of SmtB-DNA complexes. The alpha5 metal binding sites are therefore regulatory for Zn(II) sensing in vitro and in vivo, while the high-affinity alpha3N sites play some other role. This finding for SmtB is the opposite of that previously determined for Staphylococcus aureus pI258 CadC, a Pb(II)/Cd(II)/Bi(III) sensor [Busenlehner, L. S., et al. (2002) J. Mol. Biol. 319, 685-701], thus providing insight into the origin of functional metal ion selectivity in this family of metal sensor proteins.