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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.

Antimicrobial peptides (AMPs) have been an area of great interest, due to the high selectivity of these molecules toward bacterial targets over host cells and the limited development of bacterial resistance to these molecules throughout evolution. The peptide C18G has been shown to be a selective, broad spectrum AMP with a net +8 cationic charge from seven lysine residues in the sequence. In this work, the cationic Lys residues were replaced with other natural or non-proteinogenic cationic amino acids: arginine, histidine, ornithine, or diaminopropionic acid. These changes vary in the structure of the amino acid side chain, the identity of the cationic moiety, and the pKa of the cationic group. Using a combination of spectroscopic and microbiological methods, the influence of these cationic groups on membrane binding, secondary structure, and antibacterial activity was investigated. The replacement of Lys with most other cationic residues had, at most, 2-fold effects on minimal inhibitory concentration against a variety of Gram-positive and Gram-negative bacteria. However, the peptide containing His as the cationic group showed dramatically reduced activity. All peptide variants retained the ability to bind lipid vesicles and showed clear preference for binding vesicles that contained anionic lipids. Similarly, all peptides adopted a helical conformation when bound to lipids or membrane mimetics, although the peptide containing diaminopropionic acid exhibited a decreased helicity. The peptides exhibited a wider variety of activity in the permeabilization of bacterial membranes, with peptides containing Lys, Arg, or Orn being the most broadly active. In all, the antibacterial activity of the C18G peptide is generally tolerant to changes in the structure and identity of the cationic amino acids, yielding new possibilities for design and development of AMPs that may be less susceptible to immune and bacterial recognition or in vivo degradation.

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, β-(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 β-(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 β-(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 β-(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 β-(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 β-(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.

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, β-(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 β-(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 β-(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 β-(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 β-(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 β-(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.

The natural product curcumin has been shown to play a role in preventing Aβ 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 Aβ protein. To investigate these different roles, curcumin complexation to Cu(2+) was investigated in the presence and absence of two different segments of the Aβ protein including the copper-binding (Aβ6-14) and curcumin-binding (Aβ14-23) 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 Aβ14-23 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 Aβ14-23 peptide segment.

Antimicrobial peptides are short, cationic, amphiphilic naturally occurring sequences that have selective antimicrobial properties. The proposed mechanism of action for these peptides is through interaction with and disruption of the bacterial membrane. We have investigated the length and amino acid composition dependence of the antimicrobial peptides ponericin L1 and C18G on antimicrobial activity and membrane binding ability. Truncation of the peptide sequence at different points through synthesis allowed for investigation of length, overall hydrophobicity, and net charge on function. Circular dichroism and fluorescence spectroscopy were used to determine the binding affinity and structure of the peptide in the presence of lipid vesicles. All of the L1 derived peptides exhibited the ability to form alpha helices and bind to the lipid membranes to different degrees. The data suggests that the modified peptide (L1A) and the truncated peptides (L1A-13T, L1A-16T, and L1A-21T) work by forming an alpha helix to permeabilize the bacterial membrane and cause bacteriolysis. Alternatively, the C18G derived truncates exhibited a length threshold in their ability to bind membranes with high affinity and form helical structures. Ongoing experiments are probing membrane topography.

Antimicrobial peptides are naturally occurring components of the innate immune system which serve as a first line of defense against bacterial infection. While hundreds of antimicrobial peptides have been identified from natural sources, there is little in the way of consensus sequences that can identify peptides as antimicrobial. Instead, these peptides are more easily defined by the chemical properties of amphiphilicity, cationic net charge, and short length (10-30 aa). In an attempt to further elucidate the chemical mechanisms that play a role in antimicrobial activity, we compared two template peptides (C18G derived from human platelet factor and Ponericin L1 derived from ponerine ant venom) which contain arginine as the cationic moieties to the analgous pair which contain 2 - amino - 3 - guanidinopropionic acid. This effectively shortens the side chain length without modifying the cationic identity. We characterized these systems using a combination of CD spectroscopy for structure formation, fluorescence spectroscopy and quenching for lipid binding affinity, and microbiological techniques to investigate antibacterial efficacy. We found that the arginine analogs behaved similar to the parent peptides with respect to secondary structure formation in the presence of lipid bilayers, ability to permeabilize bacterial membranes, and ability to bind to anionic bilayers with high affinity. The same experiments were carried out on the 2 - amino - 3 - guanidinopropionic acid analogs. Overall this approach allows for SAR-like investigations into peptide structure/function relationships using natural and non-natural amino acids.