Impacts of Hydrophobic Mismatch on Antimicrobial Peptide Efficacy and Bilayer Permeabilization.

Antimicrobial resistance continues to be a major threat to world health, with the continued emergence of resistant bacterial strains. Antimicrobial peptides have emerged as an attractive option for the development of novel antimicrobial compounds in part due to their ubiquity in nature and the general lack of resistance development to this class of molecules. In this work, we analyzed the antimicrobial peptide C18G and several truncated forms for efficacy and the underlying mechanistic effects of the sequence truncation. The peptides were screened for antimicrobial efficacy against several standard laboratory strains, and further analyzed using fluorescence spectroscopy to evaluate binding to model lipid membranes and bilayer disruption. The results show a clear correlation between the length of the peptide and the antimicrobial efficacy. Furthermore, there is a correlation between peptide length and the hydrophobic thickness of the bilayer, indicating that hydrophobic mismatch is likely a contributing factor to the loss of efficacy in shorter peptides.

A nitric oxide synthase-like protein from Synechococcus produces NO/NO3- from l-arginine and NADPH in a tetrahydrobiopterin- and Ca2+-dependent manner.

Nitric oxide synthases (NOSs) are heme-based monooxygenases that convert l-Arg to l-citrulline and nitric oxide (NO), a key signaling molecule and cytotoxic agent in mammals. Bacteria also contain NOS proteins, but the role of NO production within these organisms, where understood, differs considerably from that of mammals. For example, a NOS protein in the marine cyanobacterium Synechococcus sp. PCC 7335 (syNOS) has recently been proposed to function in nitrogen assimilation from l-Arg. syNOS retains the oxygenase (NOSox) and reductase (NOSred) domains present in mammalian NOS enzymes (mNOSs), but also contains an N-terminal globin domain (NOSg) homologous to bacterial flavohemoglobin proteins. Herein, we show that syNOS functions as a dimer and produces NO from l-Arg and NADPH in a tetrahydrobiopterin (H4B)-dependent manner at levels similar to those produced by other NOSs but does not require Ca2+-calmodulin, which regulates NOSred-mediated NOSox reduction in mNOSs. Unlike other bacterial NOSs, syNOS cannot function with tetrahydrofolate and requires high Ca2+ levels (>200 µm) for its activation. NOSg converts NO to NO3- in the presence of O2 and NADPH; however, NOSg did not protect Escherichia coli strains against nitrosative stress, even in a mutant devoid of NO-protective flavohemoglobin. We also found that syNOS does not have NOS activity in E. coli (which lacks H4B) and that the recombinant protein does not confer growth advantages on l-Arg as a nitrogen source. Our findings indicate that syNOS has both NOS and NO oxygenase activities, requires H4B, and may play a role in Ca2+-mediated signaling.

Functional characterization of a melittin analog containing a non-natural tryptophan analog.

Tryptophan (Trp) is a naturally occurring amino acid, which exhibits fluorescence emission properties that are dependent on the polarity of the local environment around the Trp side chain. However, this sensitivity also complicates interpretation of fluorescence emission data. A non-natural analogue of tryptophan, ß-(1-azulenyl)-L-alanine, exhibits fluorescence insensitive to local solvent polarity and does not impact the structure or characteristics of several peptides examined. In this study, we investigated the effect of replacing Trp with ß-(1-azulenyl)-L-alanine in the well-known bee-venom peptide melittin. This peptide provides a model framework for investigating the impact of replacing Trp with ß-(1-azulenyl)-L-alanine in a functional peptide system that undergoes significant shifts in Trp fluorescence emission upon binding to lipid bilayers. Microbiological methods including assessment of the antimicrobial activity by minimal inhibitory concentration (MIC) assays and bacterial membrane permeability assays indicated little difference between the Trp and the ß-(1-azulenyl)-L-alanine-substituted versions of melittin. Circular dichroism spectroscopy showed both that peptides adopted the expected α-helical structures when bound to phospholipid bilayers and electrophysiological analysis indicated that both created membrane disruptions leading to significant conductance increases across model membranes. Both peptides exhibited a marked protection of the respective fluorophores when bound to bilayers indicating a similar membrane-bound topology. As expected, while fluorescence quenching and CD indicate the peptides are stably bound to lipid vesicles, the peptide containing ß-(1-azulenyl)-L-alanine exhibited no fluorescence emission shift upon binding while the natural Trp exhibited >10 nm shift in emission spectrum barycenter. Taken together, the ß-(1-azulenyl)-L-alanine can serve as a solvent insensitive alternative to Trp that does not have significant impacts on structure or function of membrane interacting peptides.

Complexation between Cu(II) and curcumin in the presence of two different segments of amyloid ß.

The natural product curcumin has been shown to play a role in preventing Aß amyloid fibril formation. This role could include chelation of transition metal ions such as Cu(2+), known to accelerate amyloid aggregation, and/or curcumin-binding directly to the Aß protein. To investigate these different roles, curcumin complexation to Cu(2+) was investigated in the presence and absence of two different segments of the Aß protein including the copper-binding (Aß6-14) and curcumin-binding (Aß14-23) domains. Absorbance and fluorescence spectroscopy in 90% water/10% methanol solutions showed that curcumin can bind Cu(2+) to some extent in the presence of both segments despite strong peptide-ion interactions. Estimated Cu(2+)-curcumin binding affinities in the absence (1.6×10(5)M(-1)) and presence (7.9×10(4)M(-1)) of the peptide provide quantitative support for this Cu(2+) chelation role. With the Aß14-23 segment, the curcumin simultaneously binds to Cu(2+) and the peptide, demonstrating that it can play multiple roles in the prevention of amyloid formation. The stabilities of ternary peptide-Cu(2+)-curcumin complexes were evaluated using ESI mass spectrometry and support the conclusion that curcumin can act as a weak metal ion chelator and also bind directly to the Aß14-23 peptide segment.