Metal ion-dependent, organophosphate-degrading enzymes have acquired increasing attention due to their ability to degrade and thus detoxify commonly used pesticides and nerve agents such as sarin. The best characterized of these enzymes are from Pseudomonas diminuta (OPH) and Agrobacterium radiobacter (OpdA). Despite high sequence homology (>90 % identity) and conserved metal ion coordination these enzymes display considerable variations in substrate specificity, metal ion affinity/preference and reaction mechanism. In this study, we highlight the significance of the presence (OpdA) or absence (OPH) of an extended hydrogen bond network in the active site of these enzymes for the modulation of their catalytic properties. In particular, the second coordination sphere residue in position 254 (Arg in OpdA, His in OPH) is identified as a crucial factor in modulating the substrate preference and binding of these enzymes. Inhibition studies with fluoride also support a mechanism for OpdA whereby the identity of the hydrolysis-initiating nucleophile changes as the pH is altered. The same is not observed for OPH.

The thermodynamics of formation of the insulin hexamer, which is stabilized by two Zn(2+) ions, were quantified by isothermal titration calorimetry (ITC). Because the insulin monomer is unstable to aggregation (fibrillation) during ITC measurements, an original method involving EDTA chelation of Zn(2+) from the hexamer was employed. The two metal ions are chelated sequentially, reflecting stepwise Zn(2+) binding and stabilization of the quaternary structure. Analysis of the ITC data reveals that two to three H(+) bind to the hexamer upon its formation at pH 7.4, which is both enthalpically and entropically favored. The former is due to Zn(2+) coordination to His residues from three subunits, and the latter is associated with desolvation that accompanies the protonation and the packing of the subunits in the hexamer.

Highlights d DNA + protein vaccine includes six sequentially isolated patient CH505 HIV Envs d DNA + Env protein co-administration in the same muscle reduces SHIV infection risk d Protection from infection is mediated by non-neutralizing antibody functions d Antibody binding to FcgRIIIa and ADCC activity contributes to protection

Zinc (Zn 2+ ) is an integral component of many proteins and has been shown to act in a regulatory capacity in different mammalian systems, including as a neurotransmitter in neurons throughout the brain. While Zn 2+ plays an important role in modulating neuronal potentiation and synaptic plasticity, little is known about the signaling mechanisms of this regulation. In dissociated rat hippocampal neuron cultures, we used fluorescent Zn 2+ sensors to rigorously define resting Zn 2+ levels and stimulation-dependent intracellular Zn 2+ dynamics, and we performed RNA-Seq to characterize Zn 2+ -dependent transcriptional effects upon stimulation. We found that relatively small changes in cytosolic Zn 2+ during stimulation altered expression levels of 931 genes, and these Zn 2+ dynamics induced transcription of many genes implicated in neurite expansion and synaptic growth. Additionally, while we were unable to verify the presence of synaptic Zn 2+ in these cultures, we did detect the synaptic vesicle Zn 2+ transporter ZnT3 and found it to be substantially upregulated by cytosolic Zn 2+ increases. These results provide the first global sequencing-based examination of Zn 2+ -dependent changes in transcription and identify genes that may mediate Zn 2+ -dependent processes and functions.

Fluorescent biosensors are important measurement tools for in vivo quantification of pH, concentrations of metal ions and other analytes, and physical parameters such as membrane potential. Both the development of these sensors and their implementation in examining cellular heterogeneity requires technology for measuring and sorting cells based on the fluorescence levels before and after chemical or physical perturbations. We developed a droplet microfluidic platform for the screening and separation of cell populations on the basis of the in vivo response of expressed fluorescence-based biosensors after addition of an exogenous analyte. We demonstrate the capability to resolve the responses of two genetically-encoded Zn2+ sensors at a range of time points spanning several seconds and subsequently sort a mixed-cell population of varying ratios with high accuracy.