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Photo-initiated rupture of azobenzene micelles to enable the spectroscopic analysis of antimicrobial peptide dynamics.

ABSTRACT: Antimicrobial peptides (AMPs) show promise for the treatment of bacterial infections, but many have undesired hemolytic activities. The AMP MP1 not only has broad spectrum bactericidal activity, but has been shown to have antitumor activity. The interaction between AMPs and cellular membranes gives rise to a peptide's cell-specificity and activity. However, direct analysis of the biophysical interactions between peptides and membrane is complex, in part due to the nature of membrane environments as well as structural changes in the peptide that occurs upon binding to the membrane. In order to investigate the interplay between cell selectivity, activity, and secondary structural changes involved in antimicrobial peptide activity, we sought to implement photolizable membrane mimics to assess the types of information available from infrared spectroscopic measurements that follow from photoinitiated peptide dynamics. Azo-surfactants (APEG) form micelles containing a photolizable azobenzene core, which upon irradiation can induce membrane deformation resulting in breakdown of micelles. Spectroscopic analysis of membrane deformation may provide insights into the physical behavior associated with unfolding and dissociation of antimicrobial peptides within a membrane environment. Herein, we synthesized and characterized two new azo-surfactants, APEG_TMG and APEG_NEt₂MeI. Furthermore, we demonstrate the viability of azosurfactants as membrane mimics by examining both the membrane binding and dissociation induced secondary structural changes of the antimicrobial peptide, MP1.

SUBMITTER: Roberson MG

PROVIDER: S-EPMC7449587 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Photo-initiated rupture of azobenzene micelles to enable the spectroscopic analysis of antimicrobial peptide dynamics.

Roberson Matthew G MG Duncan Julia M JM Flieth Keveen J KJ Geary Laina M LM Tucker Matthew J MJ

RSC advances 20200604 36

Antimicrobial peptides (AMPs) show promise for the treatment of bacterial infections, but many have undesired hemolytic activities. The AMP MP1 not only has broad spectrum bactericidal activity, but has been shown to have antitumor activity. The interaction between AMPs and cellular membranes gives rise to a peptide's cell-specificity and activity. However, direct analysis of the biophysical interactions between peptides and membrane is complex, in part due to the nature of membrane environments ...[more]

PMID: 32879729

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Project description:BACKGROUND AND PURPOSE:The photo-isomerizable local anaesthetic, quaternary ammonium-azobenzene-quaternary ammonium (QAQ), provides rapid, optical control over pain signalling without involving genetic modification. In darkness or in green light, trans-QAQ blocks voltage-gated K+ and Na+ channels and silences action potentials in pain-sensing neurons. Upon photo-isomerization to cis with near UV light, QAQ blockade is rapidly relieved, restoring neuronal activity. However, the molecular mechanism of cis and trans QAQ blockade is not known. Moreover, the absorption spectrum of QAQ requires UV light for photo-control, precluding use deep inside neural tissue. EXPERIMENTAL APPROACH:Electrophysiology and molecular modelling were used to characterize the binding of cis and trans QAQ to voltage-gated K+ channels and to develop quaternary ammonium-ethylamine-azobenzene-quaternary ammonium (QENAQ), a red-shifted QAQ derivative controlled with visible light. KEY RESULTS:trans QAQ was sixfold more potent than cis QAQ, in blocking current through Shaker K+ channels. Both isomers were use-dependent, open channel blockers, binding from the cytoplasmic side, but only trans QAQ block was slightly voltage dependent. QENAQ also blocked native K+ and Na+ channels preferentially in the trans state. QENAQ was photo-isomerized to cis with blue light and spontaneously reverted to trans within seconds in darkness, enabling rapid photo-control of action potentials in sensory neurons. CONCLUSIONS AND IMPLICATIONS:Light-switchable local anaesthetics provide a means to non-invasively photo-control pain signalling with high selectivity and fast kinetics. Understanding the mode of action of QAQ and related compounds will help to design of drugs with improved photo-pharmacological properties. LINKED ARTICLES:This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

Project description:The coordination of protein synthesis and degradation regulating protein abundance is a fundamental process in cellular homeostasis. Today, mass spectrometry-based technologies allow determination of endogenous protein turnover on a proteome-wide scale. However, standard dynamic SILAC (Stable Isotope Labeling in Cell Culture) approaches can suffer from missing data across pulse time-points limiting the accuracy of such analysis. This issue is of particular relevance when studying protein stability at the level of proteoforms because often only single peptides distinguish between different protein products of the same gene. To address this shortcoming, we evaluated the merits of combining dynamic SILAC and tandem mass tag (TMT)-labeling of ten pulse time-points in a single experiment. Although the comparison to the standard dynamic SILAC method showed a high concordance of protein turnover rates, the pulsed SILAC-TMT approach yielded more comprehensive data (6000 proteins on average) without missing values. Replicate analysis further established that the same reproducibility of turnover rate determination can be obtained for peptides and proteins facilitating proteoform resolved investigation of protein stability. We provide several examples of differentially turned over splice variants and show that post-translational modifications can affect cellular protein half-lives. For example, N-terminally processed peptides exhibited both faster and slower turnover behavior compared with other peptides of the same protein. In addition, the suspected proteolytic processing of the fusion protein FAU was substantiated by measuring vastly different stabilities of the cleavage products. Furthermore, differential peptide turnover suggested a previously unknown mechanism of activity regulation by post-translational destabilization of cathepsin D as well as the DNA helicase BLM. Finally, our comprehensive data set facilitated a detailed evaluation of the impact of protein properties and functions on protein stability in steady-state cells and uncovered that the high turnover of respiratory chain complex I proteins might be explained by oxidative stress.

Dataset Information

Photo-initiated rupture of azobenzene micelles to enable the spectroscopic analysis of antimicrobial peptide dynamics.

Publications

Photo-initiated rupture of azobenzene micelles to enable the spectroscopic analysis of antimicrobial peptide dynamics.

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