Heme Dissociation from Myoglobin in the Presence of the Zwitterionic Detergent N,N-Dimethyl-N-Dodecylglycine Betaine: Effects of Ionic Liquids

We have investigated myoglobin protein denaturation using the zwitterionic detergent Empigen BB (EBB, N,N-Dimethyl-N-dodecylglycine betaine). A combination of absorbance, fluorescence, and circular dichroism spectroscopic measurements elucidated the protein denaturation and heme dissociation from myoglobin. The results indicated that Empigen BB was not able to fully denature the myoglobin structure, but apparently can induce the dissociation of the heme group from the protein. This provides a way to estimate the heme binding free energy, ΔGdissociation. As ionic liquids (ILs) have been shown to perturb the myoglobin protein, we have investigated the effects of the ILs 1-butyl-3-methylimidazolium chloride (BMICl), 1-ethyl-3-methylimidazolium acetate (EMIAc), and 1-butyl-3-methylimidazolium tetrafluoroborate (BMIBF4) in aqueous solution on the ΔGdissociation values. Absorbance experiments show the ILs had minimal effect on ΔGdissociation values when compared to controls. Fluorescence and circular dichroism data confirm the ILs have no effect on heme dissociation, demonstrating that low concentrations ILs do not impact the heme dissociation from the protein and do not significantly denature myoglobin on their own or in combination with EBB. These results provide important data for future studies of the mechanism of IL-mediated protein stabilization/destabilization and biocompatibility studies

Effects of Ionic Liquids on Metalloproteins.

In the past decade, innovative protein therapies and bio-similar industries have grown rapidly. Additionally, ionic liquids (ILs) have been an area of great interest and rapid development in industrial processes over a similar timeline. Therefore, there is a pressing need to understand the structure and function of proteins in novel environments with ILs. Understanding the short-term and long-term stability of protein molecules in IL formulations will be key to using ILs for protein technologies. Similarly, ILs have been investigated as part of therapeutic delivery systems and implicated in numerous studies in which ILs impact the activity and/or stability of protein molecules. Notably, many of the proteins used in industrial applications are involved in redox chemistry, and thus often contain metal ions or metal-associated cofactors. In this review article, we focus on the current understanding of protein structure-function relationship in the presence of ILs, specifically focusing on the effect of ILs on metal containing proteins

Beneficial Impacts of Incorporating the Non-Natural Amino Acid Azulenyl-Alanine into the Trp-Rich Antimicrobial Peptide buCATHL4B.

Antimicrobial peptides (AMPs) present a promising scaffold for the development of potent antimicrobial agents. Substitution of tryptophan by non-natural amino acid Azulenyl-Alanine (AzAla) would allow studying the mechanism of action of AMPs by using unique properties of this amino acid, such as ability to be excited separately from tryptophan in a multi-Trp AMPs and environmental insensitivity. In this work, we investigate the effect of Trp→AzAla substitution in antimicrobial peptide buCATHL4B (contains three Trp side chains). We found that antimicrobial and bactericidal activity of the original peptide was preserved, while cytocompatibility with human cells and proteolytic stability was improved. We envision that AzAla will find applications as a tool for studies of the mechanism of action of AMPs. In addition, incorporation of this non-natural amino acid into AMP sequences could enhance their application properties

A Recurrent Silent Mutation Implicates fecA in Ethanol Tolerance by Escherichia coli.

BACKGROUND: An issue associated with efficient bioethanol production is the fact that the desired product is toxic to the biocatalyst. Among other effects, ethanol has previously been found to influence the membrane of E. coli in a dose-dependent manner and induce changes in the lipid composition of the plasma membrane. We describe here the characterization of a collection of ethanol-tolerant strains derived from the ethanologenic Escherichia coli strain FBR5. RESULTS: Membrane permeability assays indicate that many of the strains in the collection have alterations in membrane permeability and/or responsiveness of the membrane to environmental changes such as temperature shifts or ethanol exposure. However, analysis of the strains by gas chromatography and mass spectrometry revealed no qualitative changes in the acyl chain composition of membrane lipids in response to ethanol or temperature. To determine whether these strains contain any mutations that might contribute to ethanol tolerance or changes in membrane permeability, we sequenced the entire genome of each strain. Unexpectedly, none of the strains displayed mutations in genes known to control membrane lipid synthesis, and a few strains carried no mutations at all. Interestingly, we found that four independently-isolated strains acquired an identical C → A (V244 V) silent mutation in the ferric citrate transporter gene fecA. Further, we demonstrated that either a deletion of fecA or over-expression of fecA can confer increased ethanol survival, suggesting that any misregulation of fecA expression affects the cellular response to ethanol. CONCLUSIONS: The fact that no mutations were observed in several ethanol-tolerant strains suggested that epigenetic mechanisms play a role in E. coli ethanol tolerance and membrane permeability. Our data also represent the first direct phenotypic evidence that the fecA gene plays a role in ethanol tolerance. We propose that the recurring silent mutation may exert an effect on phenotype by altering RNA-mediated regulation of fecA expression

Synergistic interactions of ionic liquids and antimicrobials improve drug efficacy.

Combinations of ionic liquids (ILs) with antimicrobial compounds have been shown to produce synergistic activities in model liposomes. In this study, imidazolium chloride-based ILs with alkyl tail length variations are combined with commercially available, small-molecule antimicrobials to examine the potential for combinatorial and synergistic antimicrobial effects on P. aeruginosa, E. coli, S. aureus, and S. cerevisiae. The effects of these treatments in a human cell culture model indicate the cytotoxic limits of ILs paired with antimicrobials. The analysis of these ILs demonstrates that the length of the alkyl chain on the IL molecule is proportional to both antimicrobial activity and cytotoxicity. Moreover, the ILs which exhibit synergy with small-molecule antibiotics appear to be acting in a membrane permeabilizing manner. Collectively, results from these experiments demonstrate an increase in antimicrobial efficacy with specific IL + antimicrobial combinations on microbial cultures while maintaining low cytotoxicity in a mammalian cell culture model

Effects of Ionic Liquid Alkyl Chain Length on Denaturation of Myoglobin by Anionic, Cationic, and Zwitterionic Detergents

The unique electrochemical properties of ionic liquids (ILs) have motivated their use as solvents for organic synthesis and green energy applications. More recently, their potential in pharmaceutical chemistry has prompted investigation into their effects on biomolecules. There is evidence that some ILs can destabilize proteins via a detergent-like manner; however, the mechanism still remains unknown. Our hypothesis is that if ILs are denaturing proteins via a detergent-like mechanism, detergent-mediated protein unfolding should be enhanced in the presence of ILs. The properties of myoglobin was examined in the presence of a zwitterionic (N,N-dimethyl-N-dodecylglycine betaine (Empigen BB®, EBB)), cationic (tetradecyltrimethylammonium bromide (TTAB)), and anionic (sodium dodecyl sulfate (SDS)) detergent as well as ILs based on alkylated imidazolium chlorides. Protein structure was measured through a combination of absorbance, fluorescence, and circular dichroism (CD) spectroscopy: absorbance and CD were used to monitor heme complexation to myoglobin, and tryptophan fluorescence quenching was used as an indicator for heme dissociation. Notably, the detergents tested did not fully denature the protein but instead resulted in loss of the heme group. At low IL concentrations, heme dissociation remained a traditional, cooperative process; at high concentrations, ILs with increased detergent-like character exhibited a more complex pattern, which is most likely attributable to micellization of the ionic liquids or direct denaturation or heme dissociation induced by the ILs. These trends were consistent across all species of detergents. 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence was further used to characterize micelle formation in aqueous solutions containing detergent and ionic liquid. The dissociation thermodynamics show that EBB- and TTAB-induced dissociation of heme is not significantly impacted by room temperature ionic liquids (RTILs), whereas SDS-induced dissociation is more dramatically impacted by all RTILs examined. Together, these results indicate a complex interaction of detergents, likely based on headgroup charge, and the active component of RTILs to influence heme dissociation and potentially protein denaturation

Silver Oxide Coatings with High Silver-Ion Elution Rates and Characterization of Bactericidal Activity.

This paper reports the synthesis and characterization of silver oxide films for use as bactericidal coatings. Synthesis parameters, dissolution/elution rate, and bactericidal efficacy are reported. Synthesis conditions were developed to create AgO, Ag₂O, or mixtures of AgO and Ag₂O on surfaces by reactive magnetron sputtering. The coatings demonstrate strong adhesion to many substrate materials and impede the growth of all bacterial strains tested. The coatings are effective in killing Escherichia coli and Staphylococcus aureus, demonstrating a clear zone-of-inhibition against bacteria growing on solid media and the ability to rapidly inhibit bacterial growth in planktonic culture. Additionally, the coatings exhibit very high elution of silver ions under conditions that mimic dynamic fluid flow ranging between 0.003 and 0.07 ppm/min depending on the media conditions. The elution of silver ions from the AgO/Ag₂O surfaces was directly impacted by the complexity of the elution media, with a reduction in elution rate when examined in complex cell culture media. Both E. coli and S. aureus were shown to bind ~1 ppm Ag⁺/mL culture. The elution of Ag⁺ resulted in no increases in mammalian cell apoptosis after 24 h exposure compared to control, but apoptotic cells increased to ~35% by 48 and 72 h of exposure. Taken together, the AgO/Ag₂O coatings described are effective in eliciting antibacterial activity and have potential for application on a wide variety of surfaces and devices

Silver Oxide Coatings with High Silver-Ion Elution Rates and Characterization of Bactericidal Activity.

This paper reports the synthesis and characterization of silver oxide films for use as bactericidal coatings. Synthesis parameters, dissolution/elution rate, and bactericidal efficacy are reported. Synthesis conditions were developed to create AgO, Ag₂O, or mixtures of AgO and Ag₂O on surfaces by reactive magnetron sputtering. The coatings demonstrate strong adhesion to many substrate materials and impede the growth of all bacterial strains tested. The coatings are effective in killing Escherichia coli and Staphylococcus aureus, demonstrating a clear zone-of-inhibition against bacteria growing on solid media and the ability to rapidly inhibit bacterial growth in planktonic culture. Additionally, the coatings exhibit very high elution of silver ions under conditions that mimic dynamic fluid flow ranging between 0.003 and 0.07 ppm/min depending on the media conditions. The elution of silver ions from the AgO/Ag₂O surfaces was directly impacted by the complexity of the elution media, with a reduction in elution rate when examined in complex cell culture media. Both E. coli and S. aureus were shown to bind ~1 ppm Ag⁺/mL culture. The elution of Ag⁺ resulted in no increases in mammalian cell apoptosis after 24 h exposure compared to control, but apoptotic cells increased to ~35% by 48 and 72 h of exposure. Taken together, the AgO/Ag₂O coatings described are effective in eliciting antibacterial activity and have potential for application on a wide variety of surfaces and devices

Morphological Differentiation May Mediate Mate-Choice between Incipient Species of Anopheles gambiae s.s.

The M and S molecular forms of Anopheles gambiae s.s. have been considered incipient species for more than ten years, yet the mechanism underlying assortative mating of these incipient species has remained elusive. The discovery of the importance of harmonic convergence of wing beat frequency in mosquito mating and its relation to wing size have laid the foundation for exploring phenotypic divergence in wing size of wild populations of the two forms. In this study, wings from field collected mosquitoes were measured for wing length and wing width from two parts of the sympatric distribution, which differ with respect to the strength of assortative mating. In Mali, where assortative mating is strong, as evidenced by low rates of hybridization, mean wing lengths and wing widths were significantly larger than those from Guinea-Bissau. In addition, mean wing widths in Mali were significantly different between molecular forms. In Guinea-Bissau, assortative mating appears comparatively reduced and wing lengths and widths did not differ significantly between molecular forms. The data presented in this study support the hypothesis that wing beat frequency may mediate assortative mating in the incipient species of A. gambiae and represent the first documentation of a morphological difference between the M and S molecular forms