title: Functional characterization of a melittin analog containing a non-natural tryptophan analog.
abstract: 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.
authors: Department of Chemistry and Biochemistry, Rowan University, Glassboro NJ 08028.
authors: Zachary
authors: Z
authors: Ridgway
authors: Department of Chemistry and Biochemistry, Rowan University, Glassboro NJ 08028.
authors: Angela L
authors: AL
authors: Picciano
authors: Department of Chemistry, Syracuse University, Syracuse NY 13244.
authors: Pallavi M
authors: PM
authors: Gosavi
authors: Department of Chemistry, Syracuse University, Syracuse NY 13244.|Present affiliation: ChemBioCenter, Kyiv National Taras Shevchenko University, 61 Chervonotkatska Street, Kyiv 02094, Ukraine.
authors: Yurii S
authors: YS
authors: Moroz
authors: Department of Physics, Virginia Commonwealth University, Richmond VA 23284.
authors: Christopher E
authors: CE
authors: Angevine
authors: Department of Physics, Virginia Commonwealth University, Richmond VA 23284.
authors: Amy E
authors: AE
authors: Chavis
authors: Department of Physics, Virginia Commonwealth University, Richmond VA 23284.
authors: Joseph E
authors: JE
authors: Reiner
authors: Department of Chemistry, Syracuse University, Syracuse NY 13244.
authors: Ivan V
authors: IV
authors: Korendovych
authors: Department of Chemistry and Biochemistry, Rowan University, Glassboro NJ 08028.|School of Biomedical Sciences, Rowan University, Glassboro NJ, 08028.
authors: Gregory A
authors: GA
authors: Caputo
mesh_terms: D005453:Fluorescence; D008051:Lipid Bilayers / Q000737:chemistry*; D008555:Melitten / Q000031:analogs & derivatives / Q000737:chemistry; D017433:Protein Structure, Secondary; D014364:Tryptophan / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: D008051:Lipid Bilayers; D008555:Melitten; D014364:Tryptophan
keywords: antimicrobial peptides; azulene; fluorescence; tryptophan analogs