Electrochemical and ligand binding studies of a de novo heme protein

Abstract Heme proteins can perform a variety of electrochemical functions. While natural heme proteins carry out particular functions selected by biological evolution, artificial heme proteins, in principle, can be tailored to suit specified technological applications. Here we describe initial characterization of the electrochemical properties of a de novo heme protein, S824C. Protein S824C is a four-helix bundle derived from a library of sequences that was designed by binary patterning of polar and nonpolar amino acids. Protein S824C was immobilized on a gold electrode and the formal potential of heme-protein complex was studied as a function of pH and ionic strength. The binding of exogenous N-donor ligands to heme/S824C was monitored by measuring shifts in the potential that occurred upon addition of various concentrations of imidazole or pyridine derivatives. The response of heme/S824C to these ligands was then compared to the response of isolated heme (without protein) to the same ligands. The observed shifts in potential depended on both the concentration and the structure of the added ligand. Small changes in structure of the ligand (e.g. pyridine versus 2-amino pyridine) produced significant shifts in the potential of the heme-protein. The observed shifts correlate to the differential binding of the N-donor molecules to the oxidized and reduced states of the heme. Further, it was observed that the electrochemical response of the buried heme in heme/S824C differed significantly from that of isolated heme. These studies demonstrate that the structure of the de novo protein modulates the binding of N-donor ligands to heme

Extracellular DNA Promotes Efficient Extracellular Electron Transfer by Pyocyanin in Pseudomonas aeruginosa Biofilms

Redox cycling of extracellular electron shuttles can enable the metabolic activity of subpopulations within multicellular bacterial biofilms that lack direct access to electron acceptors or donors. How these shuttles catalyze extracellular electron transfer (EET) within biofilms without being lost to the environment has been a long-standing question. Here, we show that phenazines mediate efficient EET through interactions with extracellular DNA (eDNA) in Pseudomonas aeruginosa biofilms. Retention of pyocyanin (PYO) and phenazine carboxamide in the biofilm matrix is facilitated by eDNA binding. In vitro, different phenazines can exchange electrons in the presence or absence of DNA and can participate directly in redox reactions through DNA. In vivo, biofilm eDNA can also support rapid electron transfer between redox active intercalators. Together, these results establish that PYO:eDNA interactions support an efficient redox cycle with rapid EET that is faster than the rate of PYO loss from the biofilm

Fluorescence-based Sensing of 2,4,6-Trinitrotoluene (TNT) Using a Multi-channeled Poly(methyl methacrylate) (PMMA) Microimmunosensor

Fluorescence immunoassays employing monoclonal antibodies directed against the explosive 2,4,6-trinitrotoluene (TNT) were conducted in a multi-channel microimmunosensor. The multi-channel microimmunosensor was prepared in poly (methyl methacrylate) (PMMA) via hot embossing from a brass molding tool. The multi-channeled microfluidic device was sol-gel coated to generate a siloxane surface that provided a scaffold for antibody immobilization. AlexaFluor-cadaverine-trinitrobenzene (AlexaFluor-Cad-TNB) was used as the reporter molecule in a displacement immunoassay. The limit of detection was 1–10 ng/mL (ppb) with a linear dynamic range that covered three orders of magnitude. In addition, antibody crossreactivity was investigated using hexahydro-1,3,5-triazine (RDX), HMX, 2,4-dinitrotoluene (DNT), 4-nitrotoluene (4-NT) and 2-amino-4,6-DNT

Towards a New Standard Model for Black Hole Accretion

We briefly review recent developments in black hole accretion disk theory, emphasizing the vital role played by magnetohydrodynamic (MHD) stresses in transporting angular momentum. The apparent universality of accretion-related outflow phenomena is a strong indicator that large-scale MHD torques facilitate vertical transport of angular momentum. This leads to an enhanced overall rate of angular momentum transport and allows accretion of matter to proceed at an interesting rate. Furthermore, we argue that when vertical transport is important, the radial structure of the accretion disk is modified at small radii and this affects the disk emission spectrum. We present a simple model demonstrating how energetic, magnetically-driven outflows modify the emergent disk emission spectrum with respect to that predicted by standard accretion disk theory. A comparison of the predicted spectra against observations of quasar spectral energy distributions suggests that mass accretion rates inferred using the standard disk model may severely underestimate their true values.Comment: To appear in the Fifth Stromlo Symposium Proceedings special issue of ApS

Extracellular DNA Promotes Efficient Extracellular Electron Transfer by Pyocyanin in Pseudomonas aeruginosa Biofilms

Redox cycling of extracellular electron shuttles can enable the metabolic activity of subpopulations within multicellular bacterial biofilms that lack direct access to electron acceptors or donors. How these shuttles catalyze extracellular electron transfer (EET) within biofilms without being lost to the environment has been a long-standing question. Here, we show that phenazines mediate efficient EET through interactions with extracellular DNA (eDNA) in Pseudomonas aeruginosa biofilms. Retention of pyocyanin (PYO) and phenazine carboxamide in the biofilm matrix is facilitated by eDNA binding. In vitro, different phenazines can exchange electrons in the presence or absence of DNA and can participate directly in redox reactions through DNA. In vivo, biofilm eDNA can also support rapid electron transfer between redox active intercalators. Together, these results establish that PYO:eDNA interactions support an efficient redox cycle with rapid EET that is faster than the rate of PYO loss from the biofilm

Web Content Analysis: Expanding the Paradigm

Are established methods of content analysis (CA) adequate to analyze web content, or should new methods be devised to address new technological developments? This chapter addresses this question by contrasting narrow and broad interpretations of the concept of web content analysis. The utility of a broad interpretation that subsumes the narrow one is then illustrated with reference to research on weblogs (blogs), a popular web format in which features of HTML documents and interactive computer-mediated communication converge. The chapter concludes by proposing an expanded Web Content Analysis (WebCA) paradigm in which insights from paradigms such as discourse analysis and social network analysis are operationalized and implemented within a general content analytic framework

Integrating Paper Chromatography with Electrochemical Detection for the Trace Analysis of TNT in Soil

We report on the development of an electrochemical probe for the trace analysis of 2,4,6-trinitrotoluene (TNT) in soil samples. The probe is a combination of graphite electrodes, filter paper, with ethylene glycol and choline chloride as the solvent/electrolyte. Square wave chromatovoltammograms show the probes have a sensitivity for TNT of 0.75 nA/ng and a limit of detection of 100 ng. In addition, by taking advantage of the inherent paper chromatography step, TNT can be separated in both time and cathodic peak potential from 4-amino-dinitrotolene co-spotted on the probe or in soil samples with the presence of methyl parathion as a possible interferent