Blue fluorescent amino acid for biological spectroscopy and microscopy

Abstract

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...

Diverged Plant Terpene Synthases Reroute the Carbocation Cyclization Path towards the Formation of Unprecedented 6/11/5 and 6/6/7/5 Sesterterpene Scaffolds

“…The majority characterized to date are from marine sponges and terrestrial fungi, with only about 80 structures reported from the plant kingdom so far 3. This group of terpenes thus represents an underexplored area of chemical space that holds considerable promise for discovery of new structurally complex and diverse compounds and potential new bioactivities 4.…”

“…Fungal STSs are bifunctional chimeric proteins that consist of a TPS and a trans ‐prenytransferase (PT) domain; the latter synthesizes geranylfarnesyl diphosphate (GFPP), the substrate for TPS‐mediated cyclization, from dimethyl allyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) 5. While STSs in plants have only a TPS domain, the genes that encode these STSs tend to be physically clustered with a PT gene in plant genomes 3, 6. This clustering phenomenon has enabled us to rapidly search available sequenced plant genomes for candidate STS genes using a customized algorithm (PlantiSMASH) designed to identify clustered biosynthetic genes in plant genomes 3, 7.…”

“…While STSs in plants have only a TPS domain, the genes that encode these STSs tend to be physically clustered with a PT gene in plant genomes 3, 6. This clustering phenomenon has enabled us to rapidly search available sequenced plant genomes for candidate STS genes using a customized algorithm (PlantiSMASH) designed to identify clustered biosynthetic genes in plant genomes 3, 7. Intriguingly, although fungal and plant STSs share about 10 % protein sequence identity and are phylogenetically distant, they can in some cases catalyze the conversion of GFPP to form very similar structures via a unified bicyclic C 12 carbocation ( A1 ), for example, (+)‐quiannulatene by fungal EvQS and (−)‐ ent ‐quiannulatene by plant AtTPS25 (Figure 1 and Figure 2).…”

“…Intriguingly, although fungal and plant STSs share about 10 % protein sequence identity and are phylogenetically distant, they can in some cases catalyze the conversion of GFPP to form very similar structures via a unified bicyclic C 12 carbocation ( A1 ), for example, (+)‐quiannulatene by fungal EvQS and (−)‐ ent ‐quiannulatene by plant AtTPS25 (Figure 1 and Figure 2). 3, 5c,5d This suggests that the folds of the active site pockets of STSs from fungi and plants might be conserved to some extent, and that the ability to catalyze such cyclization has arisen through convergent evolution. …”

“…We previously identified 19 full length candidate STS genes, of which six, which spanned three main phylogenetic clades (clade I, II, III), were functionally characterized and their dominant products shown to be “fungal‐type” sesterterpenes (Figure 1 B) 3. In this study, we focus on the functional characterization of three new TPSs (Cr089 from Capsella rubella , AtTPS17 from Arabidopsis thaliana and Br580 from Brassica rapa ), belonging to an unexplored clade (clade IV) that has diverged from the three other plant STS clades (Figure 1).…”