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.

Site‐Resolved Observation of Vibrational Energy Transfer Using a Genetically Encoded Ultrafast Heater

“…[2] Compared to other donors such as the heme cofactor, AzAla is small and thus allows for ap recise localization of the VET starting point. AzAla is pseudo-isosteric to Tr pa nd has been utilized in fluorescence studies because its signal can be separated from that of Tr p. [23][24][25] With similar quantum yields to Tr p, its emission photophysics are more simple and its photostability is superior. Ther esponse of its azide side chain can be monitored by IR spectroscopy,a nd is well separated from native protein and peptide responses.…”

“…Most of the fluorescent ncAA probes installed thus far (see the Supporting Information, Figure S1 and Table S2) certainly impose significant structural changes and steric clashes when placed in al ocal protein environment. AzAla is pseudo-isosteric to Tr pa nd has been utilized in fluorescence studies because its signal can be separated from that of Tr p. [23][24][25] With similar quantum yields to Tr p, its emission photophysics are more simple and its photostability is superior. [2,23,25] To enable these versatile applications in complex proteins, an AzAla-specific orthogonal pair needs to be developed.…”

Investigation of the structure‐activity relationship in ponericin L1 from Neoponera goeldii

“…Some of the most well studied examples include melittin, mastoparan, and pardaxin. [ 10‐14 ] The relative ease of solid‐phase peptide synthesis coupled with the high aqueous solubility of these peptides have made them a model system for investigating the biophysical processes involved in peptide‐bilayer binding interactions, pore‐formation, and helix‐formation at the bilayer water interface. [ 15‐17 ] These synthetically accessible systems are also amenable to incorporation of fluorescent or other spectroscopic probes as well as nonnatural amino acids, further expanding the utility as a biophysical model system.…”

Minimalist IR and fluorescence probes of protein function

“…Incorporation of AzAla into smooth muscle myosin light chain kinase fragment did not perturb its interaction with calmodulin (CaM), moreover weak quenching of AzAla fluorescence by the methionines present in CaM allows for easy and straightforward determination of binding stoichiometry and K d for this interaction [5]. Substitution of the indole by azulene does not influence the functional properties of antimicrobial peptides: the native and AzAla-substituted melittin show essentially identical CD spectra in the presence and in the absence of lipid bilayer and have very similar MIC values in bacterial inner and outer membrane permeabilization assays [6]. Moreover, electrophysiological characterization of the peptide-lipid bilayer interaction displayed a similar pore forming kinetics for both peptides.…”