Willingness to pay for safe drinking water: A contingent valuation study in Jacksonville, FL.

A surprising number of U.S. cities have drinking water with unhealthy levels of chemicals and contaminants. The city of Jacksonville (Florida), the location for this study, owns the dubious distinction of being ranked among the worst major American cities in water quality according to water quality tests conducted between 2005 and 2009 by the Environmental Working Group (EWG). This report of toxic chemicals in the Jacksonville water supply generated considerable negative publicity and coincides with a frequent and common complaint among residents of foul-smelling water. System revenues from water supply and program subsidies from government are often inadequate in mitigating the problems, perceived or real, with water quality. Therefore, this paper investigates how much residents will be willing to pay for improvements in the quality of tap water. The commonly known economic metric willingness-to-pay (WTP) is applied to estimate any possible rate hikes public utility can assess in any effort to improve real or perceived water quality. The study shows that the estimated weighted average of WTP is $6.22, which can be added to the regular water bill without eliciting much negative reaction from residents. Evidence shows that factors such as trust in authorities, health concerns, family structure, and education significantly impact the WTP.

Micelle bound structure and DNA interaction of brevinin-2-related peptide, an antimicrobial peptide derived from frog skin.

Brevinin-2-related peptide (BR-II), a novel antimicrobial peptide isolated from the skin of frog, Rana septentrionalis, shows a broad spectrum of antimicrobial activity with low haemolytic activity. It has also been shown to have antiviral activity, specifically to protect cells from infection by HIV-1. To understand the active conformation of the BR-II peptide in membranes, we have investigated the interaction of BR-II with the prokaryotic and eukaryotic membrane-mimetic micelles such as sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC), respectively. The interactions were studied using fluorescence and circular dichroism (CD) spectroscopy. Fluorescence experiments revealed that the N-terminus tryptophan residue of BR-II interacts with the hydrophobic core of the membrane mimicking micelles. The CD results suggest that interactions with membrane-mimetic micelles induce an α-helix conformation in BR-II. We have also determined the solution structures of BR-II in DPC and SDS micelles using NMR spectroscopy. The structural comparison of BR-II in the presence of SDS and DPC micelles showed significant conformational changes in the residues connecting the N-terminus and C-terminus helices. The ability of BR-II to bind DNA was elucidated by agarose gel retardation and fluorescence experiments. The structural differences of BR-II in zwitterionic versus anionic membrane mimics and the DNA binding ability of BR-II collectively contribute to the general understanding of the pharmacological specificity of this peptide towards prokaryotic and eukaryotic membranes and provide insights into its overall antimicrobial mechanism.

Solution structures and model membrane interactions of Ctriporin, an anti-methicillin-resistant Staphylococcus aureus Peptide from Scorpion Venom.

Ctriporin peptide (Ctr), a novel antimicrobial peptide isolated from the venom of the scorpion Chaerilus tricostatus, shows a broad-spectrum of antimicrobial activity and is able to inhibit antibiotic resistant pathogens, including Methicillin resistant Staphylococcus aureus, Methicillin Resistant Coagulase-negative Staphylococcus, and Penicillin Resistant Staphylococcus epidermidis strains. To understand the active conformation of the Ctr peptide in membranes, we have investigated the interaction of Ctr with the negatively charged and zwitterionic membrane-mimetic micelles such as sodium dodecyl sulphate (SDS) and n-dodecylphosphocholine (DPC), respectively. The interactions were studied using fluorescence and circular dichroism (CD) spectroscopy. Fluorescence experiments revealed that the N-terminus tryptophan residue of Ctr interacted with the hydrophobic core of the membrane mimicking micelles. The CD results suggest that interactions with membrane-mimetic micelles induce an α-helix conformation in Ctr. Moreover, we have determined the solution structures of Ctr in SDS and DPC micelles using nuclear magnetic resonance (NMR) spectroscopy. The structural comparison of Ctr in the presence of SDS and DPC micelles showed significant conformational changes. The observed structural differences of Ctr in anionic versus zwitterionic membrane-mimetic micelles suggest that the mode of interaction of this peptide may be different in two environments which may account for its ability to differentiate bacterial and eukaryotic cell membrane.

Sequence context induced antimicrobial activity: insight into lipopolysaccharide permeabilization.

Lactoferrampin (WR17, Trp 268-Arg 284), an antimicrobial peptide, is known to have significant antibacterial and candidacidal activities. However, there are no previous studies explaining how WR17 permeabilizes the outer membrane of Gram negative bacteria and neutralizes endotoxins. In this study we used a series of assays like antimicrobial activity, calcein leakage, NPN dye uptake and endotoxin neutralization assay to show that the sequence context of WR17 modulates its multi-faceted activities. We determined the high resolution NMR structure of WR17 in LPS and found that the N-ter region forms a helix (Trp1-Phe11) and orients itself at an angle of 45° into the lipopolysaccharide (LPS) micelle, whereas the C-ter region (Lys13-Arg17) remains as a flexible extended random coil. We also verified this result through in silico molecular modeling simulation. Isothermal titration calorimetry showed that the interaction of WR17 and its analogues with LPS was primarily endothermic in nature. Using several fluorescence techniques such as anisotropy and red edge excitation shift assay we revealed motional restriction for Trp1 of WR17 in LPS. The distance between the indole ring of Trp1 of WR17 and the polar head group of LPS is around 7 Å, as obtained from the depth of insertion assay. Additionally, MD simulation demonstrated that the incorporation of the peptide in LPS is achieved with the help of the K(13)xK(15)xR(17) motif at the C-terminus. This novel anchoring "K(13)NKSR(17)" motif is currently being utilized in our ongoing research to design novel anti-endotoxic molecules.

Understanding household preferences for hurricane risk mitigation information: evidence from survey responses.

Risk information is critical to adopting mitigation measures, and seeking risk information is influenced by a variety of factors. An essential component of the recently adopted My Safe Florida Home (MSFH) program by the State of Florida is to provide homeowners with pertinent risk information to facilitate hurricane risk mitigation activities. We develop an analytical framework to understand household preferences for hurricane risk mitigation information through allowing an intensive home inspection. An empirical analysis is used to identify major drivers of household preferences to receive personalized information regarding recommended hurricane risk mitigation measures. A variety of empirical specifications show that households with home insurance, prior experience with damages, and with a higher sense of vulnerability to be affected by hurricanes are more likely to allow inspection to seek information. However, households with more members living in the home and households who live in manufactured/mobile homes are less likely to allow inspection. While findings imply MSFH program's ability to link incentives offered by private and public agencies in promoting mitigation, households that face a disproportionately higher level of risk can get priority to make the program more effective.

Model membrane interaction and DNA-binding of antimicrobial peptide Lasioglossin II derived from bee venom.

Lasioglossins, a new family of antimicrobial peptide, have been shown to have strong antimicrobial activity with low haemo-lytic and mast cell degranulation activity, and exhibit cytotoxic activity against various cancer cells in vitro. In order to understand the active conformation of these pentadecapeptides in membranes, we have studied the interaction of Lasioglossin II (LL-II), one of the members of Lasioglossins family with membrane mimetic micelle Dodecylphosphocholine (DPC) by fluorescence, Circular Dichroism (CD) and two dimensional (2D) (1)H NMR spectroscopy. Fluorescence experiments provide evidence of interaction of the N-terminal tryptophan residue of LL-II with the hydrophobic core of DPC micelle. CD results show an extended chain conformation of LL-II in water which is converted to a partial helical conformation in the presence of DPC micelle. Moreover we have determined the first three-dimensional NMR structure of LL-II bound to DPC micelle with rmsd of 0.36Å. The solution structure of LL-II shows hydrophobic and hydrophilic core formation in peptide pointing towards different direction in the presence of DPC. This amphipathic structure may allow this peptide to penetrate deeply into the interfacial region of negatively charged membranes and leading to local membrane destabilization. Further we have elucidated the DNA binding ability of LL-II by agarose gel retardation and fluorescence quenching experiments.

Crystal structure of the heteromolecular chaperone, AscE-AscG, from the type III secretion system in Aeromonas hydrophila.

BACKGROUND: The putative needle complex subunit AscF forms a ternary complex with the chaperones AscE and AscG in the type III secretion system of Aeromonas hydrophila so as to avoid premature assembly. Previously, we demonstrated that the C-terminal region of AscG (residues 62-116) in the hetero-molecular chaperone, AscE-AscG, is disordered and susceptible to limited protease digestion. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report the crystal structure of the ordered AscG(1-61) region in complex with AscE at 2.4 Å resolution. Helices α2 and α3 of AscE in the AscE-AscG(1-61) complex assumes a helix-turn-helix conformation in an anti-parallel fashion similar to that in apo AscE. However, in the presence of AscG, an additional N-terminal helix α1 in AscE (residues 4-12) is observed. PscG or YscG in the crystal structures of PscE-PscF-PscG or YscE-YscF-YscG, respectively, assumes a typical tetratricopeptide repeat (TPR) fold with three TPR repeats and one C-terminal capping helix. By comparison, AscG in AscE-AscG(1-61) comprises three anti-parallel helices that resembles the N-terminal TPR repeats in the corresponding region of PscG or YscG in PscE-PscF-PscG or YscE-YscF-YscG. Thermal denaturation of AscE-AscG and AscE-AscG(1-61) complexes demonstrates that the C-terminal disordered region does not contribute to the thermal stability of the overall complex. CONCLUSION/SIGNIFICANCE: The N-terminal region of the AscG in the AscE-AscG complex is ordered and assumes a structure similar to those in the corresponding regions of PscE-PscG-PscF or YscE-YscF-YscG complexes. While the C-terminal region of AscG in the AscE-AscG complex is disordered and will assume its structure only in the presence of the substrate AscF. We hypothesize that AscE act as a chaperone of the chaperone to keep AscG in a stable but partially disordered state for interaction with AscF.

Temperature and Mg2+ sensing by a novel PhoP-PhoQ two-component system for regulation of virulence in Edwardsiella tarda.

The PhoP-PhoQ two-component system is commonly used by bacteria to sense environmental factors. Here we show that the PhoP-PhoQ system of Edwardsiella tarda detects changes in environmental temperature and Mg(2+) concentration as well as regulates the type III and VI secretion systems through direct activation of esrB. Protein secretion is activated from 23 to 35 °C or at low Mg(2+) concentrations, but it is suppressed at or below 20 °C, at or above 37 °C, or at high Mg(2+) concentrations. The effects of temperature and Mg(2+) concentration are additive. The PhoQ sensor domain has a low T(m) of 37.9 °C, and it detects temperatures through a conformational change of its secondary structure. Mutation of specific Pro or Thr residues increased the stability of the PhoQ sensor drastically, altering its temperature-sensing ability. The PhoQ sensor detects Mg(2+) concentration through the direct binding of Mg(2+) to a cluster of acidic residues (DDDSAD) and through changes that likely affect its tertiary structure. Here, we describe for the first time the use of PhoP-PhoQ as a temperature sensor for bacterial virulence control.

GM1-induced structural changes of bovine serum albumin after chemical and thermal disruption of the secondary structure: a spectroscopic comparison.

GM1-induced structural transitions of native and unfolded conformers of bovine serum albumin (BSA) have been studied where in the unfolded conformers, the secondary structures were disrupted either chemically by 8 M urea or thermally by heating at 65 degrees C. With decreasing protein:ganglioside ratio at pH 7.0, the native BSA partially unfolds and expands, while the urea-denatured BSA forms an alpha-helical structural pattern with shrinking in the conformational space. However, a continuous loss of alpha-helicity with minor increase in size was observed for the thermally altered protein in the presence of the GM1 micelle. The changes in the secondary structural content were followed by far-UV circular dichroism (CD) analysis. The dynamic light scattering (DLS) experiments were used to study the variation of the size of the protein-GM1 complexes with increasing concentration of the GM1. Fluorescence experiments show that tryptophan residues of BSA experience a more hydrophobic environment in the presence of the GM1 micelle with a decreasing protein:ganglioside ratio at pH 7.0. The present study shows that GM1 has a strong effect on the conformation of BSA depending on the conformational states of the protein that would relate to a physiological function of GM1 such as acting as the receptor of proteins in the cell membrane.

Interactions of trifluoroethanol with [val5]angiotensin II.

Intermolecular 1H{19F} NOE experiments have been used to explore the interactions of trifluoroethanol (TFE) with the octapeptide hormone [val5]angiotensin II at temperatures from 5 to 25 degrees C. Circular dichroism spectra indicate that 40% trifluoroethanol has an influence on the conformations of the peptide, probably leading to beta-structures. Diffusion experiments show that the mean hydrodynamic radius of the peptide in 40% trifluoroethanol-water is about 8 A, consistent with significant folding of the peptide in this medium. Distance constraints derived from intramolecular NOESY data along with observed vicinal coupling constants (3JCalphaHNH) were used to develop conformations consistent with available data. Assuming that intermolecular 1H{19F} NOEs are the result of diffusive encounters of TFE and peptide molecules, it is shown that no single conformation is consistent with the experimental values of the sigmaHF cross-relaxation parameters. It is argued that the disagreements between observed and expected values of sigmaHF are the result of formation of long-lived (approximately 0.5 ns) fluoroalcohol-peptide complexes, a conclusion consonant with similar studies of other peptide-fluoroalcohol systems. Complex formation appears to be especially prevalent near the charged amino acid side chains of the hormone.

Interactions of trifluroethanol with the Trp-cage peptide.

It has been suggested that aggregation of fluorinated alcohols in water solutions is involved with the abilities of these alcohols to provoke conformational changes in peptides and proteins. The extent of fluoroalcohol aggregation depends on the degree of fluorination: hexafluoroisopropanol (HFIP) is more extensively aggregated than is TFE. We previously described a study of the interactions of HFIP with the peptide Trp-cage and provided evidence for the formation of long-lived complexes between this fluoroalcohol and the peptide. In the present work, we have examined the interactions of the less-fluorinated TFE with Trp-cage, in order to probe the role of fluoroalcohol aggregation in the phenomena observed. Intermolecular (1)H{(19)F} nuclear Overhauser effects arising from interactions of TFE with the hydrogens of the peptide in a solution containing 42% TFE were determined at sample temperatures from 5 to 45 degrees C. It is shown that the folded state of the peptide under these conditions is essentially the same as that observed in water and in 30% HFIP-water. The observed peptide-solvent NOEs indicate formation of complexes of Trp-cage with TFE that persist for times of the order of 1 ns. The interactions leading to complexes with TFE are somewhat weaker than those involved in complex formation with HFIP. There are no indications that the aggregation of fluoroalcohol is a necessary concomitant of the interactions of TFE or HFIP with Trp-cage. Rather, the stronger and more long-lived interactions of HFIP with Trp-cage appear to be primarily the result of the greater hydrogen-bonding ability and hydrophobicity of this fluoroalcohol.

Interactions of hexafluoro-2-propanol with the Trp-cage peptide.

Fluoro alcohols present in aqueous solutions can alter the dominant conformations of peptides and proteins. The origins of these effects likely are related to the details of solute-fluoro alcohol interactions. Preferential interaction of the fluoro alcohol component of a fluoro alcohol-water mixture with peptide solutes has been demonstrated by several experimental approaches. In the present work, we have used 1H{19F} intermolecular NOE experiments to examine interactions of hexafluoro-2-propanol in a 30% fluoro alcohol-50 mM phosphate buffer solvent mixture with the "Trp-cage" peptide (NLY IQW LKD GGP SSG RPP PS). The results show that the peptide is selectively solvated by hexafluoro-2-propanol to the extent that the fluoro alcohol concentration near the peptide may be 3 to 4 times higher than the nominal concentration of fluoro alcohol in the bulk sample. The observed NOEs indicate that peptide-fluoro alcohol interactions persist for times of the order of 1 ns at 5 degrees C. As the sample temperature is increased, the lifetimes of fluoro alcohol interactions with several exposed side chains decrease to the extent that the peptide hydrogen-solvent fluorine interactions appear to become diffusive in nature, with interaction lifetimes of approximately 0.03 ns. It is known that protein molecules can provide specific sites for binding small organic solvent molecules. Our work suggests that small peptides also have this ability and that the dynamics for such interactions can be site-specific.

Binding and folding of melittin in the presence of ganglioside GM1 micelles.

In this work we examine the binding and folding of the membrane-active peptide, melittin in the presence of ganglioside GM1 micelle. The membrane bilayer is capable of inducing folding to small proteins and peptides upon binding. Using two-dimensional NMR techniques we have shown that at low concentration, GM1 micelle is able to induce an extended helical conformation to MLT. The pulsed-field gradient diffusion NMR study indicates that the peptide partition into GM1 micelle along with about 32% binding. While looking for the binding between MLT and GM1 using saturation transfer difference NMR spectroscopy, Val5, Leu9, Thr11, Ile17, Ser18, and Trp19 have been identified as the residues that are in close proximity to GM1 micelles.

Interaction and structural study of kinin peptide bradykinin and ganglioside monosialylated 1 micelle.

Partitioning of small proteins and peptides from the aqueous to membrane phase is often coupled with folding. In this work we examine the binding and folding of the kinin peptide, bradykinin (BK), in the presence of the ganglioside monosialylated 1 (GM1) micelle. Using two-dimensional NMR techniques, we have shown that at low concentration, GM1 micelle is able to induce a turn conformation to BK. A pulsed-field gradient diffusion NMR study indicated that the peptide partitions into the GM1 micelle with a DeltaG(part) of -3.14 +/- 0.03 kcal/mol. A saturation transfer difference (STD) NMR study indicated that the binding is mostly through hydrophobic residues.

Conformational alteration of bradykinin in presence of GM1 micelle.

We report here the interaction of bradykinin with ganglioside GM1 by circular dichroism, steady-state fluorescence, and one-dimensional 1H NMR spectroscopy. Circular dichroism spectroscopy is indicative of a turn formation of bradykinin backbone in the presence of GM1 micelle. The fluorescence quenching efficiencies of iodide and acrylamide are substantially reduced, indicating a shielding of phenylalanine residue of bradykinin from aqueous environment. Significant line broadening of NMR resonances of bradykinin, suggestive of motional restriction, is observed.

Structural alterations of enkephalins in the presence of GM1 ganglioside micelles.

The enkephalins are endogenous neurotransmitters and bind with high affinity at the delta-receptor. Gangliosides, the major glycans of nerve cells, known to interact both with receptors and ligands on the cell surface, have been implicated to modulate the actions of opioid receptors by allosteric regulation (Wu, G.; Lu, Z. H.; Wei, T. J.; Howells, R. D.; Christoffers, K.; Leeden R. W. Ann NY Acad Sci 1998, 845, 126-138). We have studied the interactions between enkephalins and monosialylated ganglioside GM1 using NMR spectroscopy and fluorescence. The structural models of enkephalins in the presence of GM1 micelles were generated using two-dimensional (1)H-ROESY experiments along with restrained molecular dynamics simulations. We report a conformational alteration of enkephalins in the presence of GM1 micelles.

Melittin-GM1 interaction: a model for a side-by-side complex.

We report here the interaction of melittin with ganglioside GM1 by steady-state fluorescence, one-dimensional (1)H NMR spectroscopy and molecular modeling. In the presence of GM1 the emission maximum of melittin is blue shifted and fluorescence quenching efficiencies of iodide and acrylamide are substantially reduced, indicating a shielding of tryptophan of melittin from aqueous environment. Significant line broadening of NMR resonances of melittin, suggestive of motional restriction, is observed. Molecular modeling indicates a melittin-GM1 complex with N-terminal hydrophobic stretch of melittin associating with the ceramide tail and C-terminal hydrophilic end of melittin having favorable electrostatic interaction with the carbohydrate head group of GM1.