Semiquinone and cluster N6 signals in His-tagged proton-translocating NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli.

NADH:ubiquinone oxidoreductase (complex I) pumps protons across the membrane using downhill redox energy. The Escherichia coli complex I consists of 13 different subunits named NuoA-N coded by the nuo operon. Due to the low abundance of the protein and some difficulty with the genetic manipulation of its large ~15-kb operon, purification of E. coli complex I has been technically challenging. Here, we generated a new strain in which a polyhistidine sequence was inserted upstream of nuoE in the operon. This allowed us to prepare large amounts of highly pure and active complex I by efficient affinity purification. The purified complex I contained 0.94 ± 0.1 mol of FMN, 29.0 ± 0.37 mol of iron, and 1.99 ± 0.07 mol of ubiquinone/1 mol of complex I. The extinction coefficient of isolated complex I was 495 mM(-1) cm(-1) at 274 nm and 50.3 mM(-1) cm(-1) at 410 nm. NADH:ferricyanide activity was 219 ± 9.7 µmol/min/mg by using HEPES-Bis-Tris propane, pH 7.5. Detailed EPR analyses revealed two additional iron-sulfur cluster signals, N6a and N6b, in addition to previously assigned signals. Furthermore, we found small but significant semiquinone signal(s), which have been reported only for bovine complex I. The line width was ~12 G, indicating its neutral semiquinone form. More than 90% of the semiquinone signal originated from the single entity with P½ (half-saturation power level) = 1.85 milliwatts. The semiquinone signal(s) decreased by 60% when with asimicin, a potent complex I inhibitor. The functional role of semiquinone and the EPR assignment of clusters N6a/N6b are discussed.

Multiplexed detection of viral infections using rapid in situ RNA analysis on a chip.

Viral infections are a major cause of human disease, but many require molecular assays for conclusive diagnosis. Current assays typically rely on RT-PCR or ELISA; however, these tests often have limited speed, sensitivity or specificity. Here, we demonstrate that rapid RNA FISH is a viable alternative method that could improve upon these limitations. We describe a platform beginning with software to generate RNA FISH probes both for distinguishing related strains of virus (even those different by a single base) and for capturing large numbers of strains simultaneously. Next, we present a simple fluidic device for reliably performing RNA FISH assays in an automated fashion. Finally, we describe an automated image processing pipeline to robustly identify uninfected and infected samples. Together, our results establish RNA FISH as a methodology with potential for viral point-of-care diagnostics.