Painting proteins blue: β-(1-azulenyl)-l-alanine as a probe for studying protein–protein interactions

Moroz, Yurii S.; Binder, Wolfgang H.; Nygren, Patrik; Caputo, Gregory A.; Korendovych, Ivan V.

doi:10.1039/c2cc37550h

Cited by 50 publications

(47 citation statements)

References 30 publications

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“…[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.…”

Section: Anisotropicvibrationalenergytransfer(vet)isexpectedtomentioning

confidence: 99%

“…Endowed with these unique characteristics,A zAla presents ak ey tool to study VET in proteins.C ompleting the donor-sensor pair, the ncAA l-azidohomoalanine (Aha;F igure 1a)r epresents aw ell-suited VET sensor. [2,23,25] To enable these versatile applications in complex proteins, an AzAla-specific orthogonal pair needs to be developed. [2] Aside from being the key tool for VET studies in proteins,t he demand for the incorporation of AzAla is fueled by its properties as af luorescent label.…”

Section: Anisotropicvibrationalenergytransfer(vet)isexpectedtomentioning

confidence: 99%

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

et al. 2019

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Allosteric information transfer in proteins has been linked to distinct vibrational energy transfer (VET) pathways in an umber of theoretical studies.E xperimental evidence for such pathways, however,i ss parse because site-selective injection of vibrational energy into aprotein, that is,localized heating,isrequired for their investigation. Here,wesolved this problem by the site-specific incorporation of the non-canonical amino acid b-(1-azulenyl)-l-alanine (AzAla) through genetic code expansion. As an exception to Kashasr ule,A zAla undergoes ultrafast internal conversion and heating after S 1 excitation while upon S 2 excitation, it serves as af luorescent label. We equipped PDZ3, ap rotein interaction domain of postsynaptic density protein 95, with this ultrafast heater at two distinct positions.W eindeed observed VET from the incorporation sites in the protein to ab ound peptide ligand on the picosecond timescale by ultrafast IR spectroscopy. This approach based on genetically encoded AzAla paves the way for detailed studies of VET and its role in aw ide range of proteins. Dr.J .Jaric Present address:H ospira Zagreb d.o.o.,aPfizer company Prudnicka cesta 60, 10291 Prigorje Brdovecko (Croatia) [ + + ]T hese authors contributed equally to this work. Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.

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Section: Anisotropicvibrationalenergytransfer(vet)isexpectedtomentioning

confidence: 99%

Section: Anisotropicvibrationalenergytransfer(vet)isexpectedtomentioning

confidence: 99%

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

et al. 2019

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“…Recently, we reported that β‐(1‐azulenyl)‐L‐alanine (AzAla), a minimally disruptive mimic of tryptophan (Figure ), can be successfully incorporated into calmodulin‐binding peptides without loss of function . While being a structurally conservative replacement, AzAla has a number of unique spectral and photophysical characteristics compared to Trp.…”

Section: Introductionmentioning

confidence: 99%

Functional characterization of a melittin analog containing a non‐natural tryptophan analog

et al. 2015

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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.

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“…The many tryptophan analogs reported throughout the years still possess the same properties [3,4]. Recently we demonstrated that β-(1-azulenyl)-L-alanine (AzAla), a tryptophan mimic, can serve as an excellent alternative to tryptophan [5]. Fluorescence properties of AzAla are not dependent on the local environment and allow for observation of very small effects exerted by the weak native quenchers inherently present in many proteins, such as histidine and methionine.…”

Section: Fluorescence Probesmentioning

confidence: 99%

“…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].…”

Section: Fluorescence Probesmentioning

confidence: 99%

Minimalist IR and fluorescence probes of protein function

Gosavi

Korendovych

2016

Current Opinion in Chemical Biology

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Spectroscopic studies of small proteins and peptides, especially those requiring fine spatial and/or temporal resolution, demand synthetic probes that confer the minimal possible steric and functional change on the native properties. Here we review the recent progress in development of minimally disruptive probes for fluorescence and infrared spectroscopies, as well as the methods to efficiently incorporate them into proteins. Advances in spectroscopy on the one hand result in high specialization of synthetic probes for a particular purpose, but on the other hand allow for the same probes be used for different techniques to gather complementary biochemical information.

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Painting proteins blue: β-(1-azulenyl)-l-alanine as a probe for studying protein–protein interactions

Abstract: We demonstrated that β-(1-Azulenyl)-L-Alanine, a fluorescent pseudoisosteric analog of tryptophan, exhibits weak environmental dependence and thus allows for using weak intrinsic quenchers, such as methionines, to monitor protein-protein interactions while not perturbing them.

Cited by 50 publications

References 30 publications

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

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

Functional characterization of a melittin analog containing a non‐natural tryptophan analog

Minimalist IR and fluorescence probes of protein function

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