Tryptophan-based fluorophores for studying protein conformational changes

Talukder, Poulami; Chen, Shengxi; Liu, C. Tony; Baldwin, Edwin Alexander; Benkovic, Stephen J.; Hecht, Sidney M.

doi:10.1016/j.bmc.2014.09.015

Cited by 27 publications

(29 citation statements)

References 56 publications

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“…Many past studies have focused on Trp-based unnatural amino acids, including azatryptophans (5,6,12) and various indole-ring substituted analogs (13)(14)(15)(16), aiming to identify useful biological fluorophores. Whereas some Trp analogs indeed exhibit improved fluorescent properties over Trp, none of them has found broad applications due to certain photophysical limitations.…”

mentioning

confidence: 99%

Blue fluorescent amino acid for biological spectroscopy and microscopy

Hilaire

Ahmed

Lin

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

116

119

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Many fluorescent proteins are currently available for biological spectroscopy and imaging measurements, allowing a wide range of biochemical and biophysical processes and interactions to be studied at various length scales. However, in applications where a small fluorescence reporter is required or desirable, the choice of fluorophores is rather limited. As such, continued effort has been devoted to the development of amino acid-based fluorophores that do not require a specific environment and additional time to mature and have a large fluorescence quantum yield, long fluorescence lifetime, good photostability, and an emission spectrum in the visible region. Herein, we show that a tryptophan analog, 4-cyanotryptophan, which differs from tryptophan by only two atoms, is the smallest fluorescent amino acid that meets these requirements and has great potential to enable in vitro and in vivo spectroscopic and microscopic measurements of proteins.fluorescence protein | fluorescence probe | unnatural amino acid | imaging O f the amino acids that are inherently responsible for the fluorescence of proteins, tyrosine (Tyr), tryptophan (Trp), and phenylalanine (Phe), Trp is the most widely used fluorescence reporter of protein structure, function, and dynamics, as its fluorescence quantum yield (QY) is comparatively large and is also sensitive to environment (1-3). However, Trp absorbs/emits in the UV wavelength region and has low photostability. Combined, these factors render this naturally occurring fluorescent amino acid hardly useful as a fluorophore for single-molecule measurements (4) and imaging applications, especially under in vivo conditions. For this reason, significant efforts have been devoted to identifying small unnatural fluorophores (5-11) that could overcome this limitation. So far, only naphthalene-based fluorophores, such as prodan (8, 9) and aladan (11) have been shown to be useful in this regard. However, these fluorophores are not structurally based on any naturally occurring amino acids and are larger in size than Trp, making them less attractive in applications where there are stringent requirements for the fluorophore size and structure. Therefore, the development of a smaller, ideally amino acid-based, fluorophore that does not require additional time to mature, have a large fluorescence QY, long fluorescence lifetime, good photostability, and an emission spectrum in the visible range, would enhance biological research. Herein, we show that a Trp analog, 4-cyanotryptophan (4CN-Trp), meets these requirements and has great potential to expand biological fluorescence spectroscopy and microscopy into additional territory.Many past studies have focused on Trp-based unnatural amino acids, including azatryptophans (5, 6, 12) and various indole-ring substituted analogs (13-16), aiming to identify useful biological fluorophores. Whereas some Trp analogs indeed exhibit improved fluorescent properties over Trp, none of them has found broad applications due to certain photophysical limitations. Recently, Ta...

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mentioning

confidence: 99%

Blue fluorescent amino acid for biological spectroscopy and microscopy

Hilaire

Ahmed

Lin

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

116

119

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“…Described in this report are the synthesis and photophysical properties of a library of dipeptidomimetic analogues and their ribosomal incorporation into position 66 of Aequorea victoria GFP. Selected fluorophores with interesting properties were made in greater amounts and characterized with regard to their environmental sensitivity and ability to act as a FRET donor following incorporation into the β -barrel structural domain of GFP, using acridon-2-ylalanine (Acd) 14 as the acceptor.…”

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confidence: 99%

Synthesis and Evaluation of a Library of Fluorescent Dipeptidomimetic Analogues as Substrates for Modified Bacterial Ribosomes

et al. 2016

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Described herein are the synthesis and photophysical characterization of a library of aryl-substituted oxazole- and thiazole-based dipeptidomimetic analogues, and their incorporation into position 66 of green fluorescent protein (GFP) in lieu of the natural fluorophore. These fluorescent analogues resemble the fluorophore formed naturally by GFP. As anticipated, the photophysical properties of the analogues varied as a function of the substituents at the para position of the phenyl ring. The fluorescence emission wavelength maxima of compounds in the library varied from ~365 nm (near-UV region) to ~490 nm (visible region). The compounds also exhibited a large range of quantum yields (0.01–0.92). The analogues were used to activate a suppressor tRNACUA and were incorporated into position 66 of GFP using an in vitro protein biosynthesizing system that employed engineered ribosomes selected for their ability to incorporate dipeptides. Four analogues with interesting photophysical properties and reasonable suppression yields were chosen, and the fluorescent proteins (FPs) containing these fluorophores were prepared on a larger scale for more detailed study. When the FPs were compared with the respective aminoacyl-tRNAs and the actual dipeptide analogues, the FPs exhibited significantly enhanced fluorescence intensities at the same concentrations. Part of this was shown to be due to the presence of the fluorophores as an intrinsic element of the protein backbone. There were also characteristic shifts in the emission maxima, indicating the environmental sensitivity of these probes. Acridon-2-ylalanine and oxazole 1a were incorporated into positions 39 and 66 of GFP, respectively, and were shown to form an efficient Förster resonance energy transfer (FRET) pair, demonstrating that the analogues can be used as FRET probes.

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“…Labeling proteins with high specificity and site-selectivity provided by NNAA-mediated conjugation has greatly expanded the repertoire of proteins amenable to investigation by existing analytic methods. Fluorescent resonance energy transfer (FRET) is a valuable tool to measure inter-or intra-molecular distance and its variation upon protein-protein interaction and conformational changes (Schuler & Hofmann, 2013;Talukder et al, 2014). Energy transfer efficiency between two different fluorophores is inversely proportional to the sixth power of their distance, therefore rendering it a highly sensitive tool for studying protein dynamics.…”

Section: Biochemical Analysismentioning

confidence: 99%

Bioconjugation of therapeutic proteins and enzymes using the expanded set of genetically encoded amino acids

Lim

Kwon

2015

Critical Reviews in Biotechnology

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The last decade has witnessed striking progress in the development of bioorthogonal reactions that are strictly directed towards intended sites in biomolecules while avoiding interference by a number of physical and chemical factors in biological environment. Efforts to exploit bioorthogonal reactions in protein conjugation have led to the evolution of protein translational machineries and the expansion of genetic codes that systematically incorporate a range of non-natural amino acids containing bioorthogonal groups into recombinant proteins in a site-specific manner. Chemoselective conjugation of proteins has begun to find valuable applications to previously inaccessible problems. In this review, we describe bioorthogonal reactions useful for protein conjugation, and biosynthetic methods that produce proteins amenable to those reactions through an expanded genetic code. We then provide key examples in which novel protein conjugates, generated by the genetic incorporation of a non-natural amino acid and the chemoselective reactions, address unmet needs in protein therapeutics and enzyme engineering.

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Tryptophan-based fluorophores for studying protein conformational changes

Cited by 27 publications

References 56 publications

Blue fluorescent amino acid for biological spectroscopy and microscopy

Blue fluorescent amino acid for biological spectroscopy and microscopy

Synthesis and Evaluation of a Library of Fluorescent Dipeptidomimetic Analogues as Substrates for Modified Bacterial Ribosomes

Bioconjugation of therapeutic proteins and enzymes using the expanded set of genetically encoded amino acids

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