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...

The M2 proton channel of the Influenza A virus has been the subject of extensive studies because of its critical role in viral replication. As such, we now know a great deal about its mechanism of action, especially how it selects and conducts protons in an asymmetric fashion. The conductance of this channel is tuned to conduct protons at a relatively low biologically useful rate, which allows acidification of the viral interior of a virus entrapped within an endosome, but not so great as to cause toxicity to the infected host cell prior to packaging of the virus. The dynamic, structural and chemical features that give rise to this tuning are not fully understood. Herein, we use a tryptophan (Trp) analog, 5-cyanotryptophan, and various methods, including linear and nonlinear infrared spectroscopies, static and time-resolved fluorescence techniques, and molecular dynamics simulations, to site-specifically interrogate the structure and hydration dynamics of the Trp41 gate in the transmembrane domain of the M2 proton channel. Our results suggest that the Trp41 sidechain adopts the t90 rotamer whose χ2 dihedral angle undergoes an increase of approximately 35° upon changing the pH from 7.4 to 5.0. Furthermore, we find that Trp41 is situated in an environment lacking bulk-like water, and somewhat surprisingly, the water density and dynamics do not show a measurable difference between the high (7.4) and low (5.0) pH states. Since previous studies have shown that upon channel opening water flows into the cavity above the histidine tetrad (His37), the present finding thus provides evidence indicating that the lack of sufficient water molecules near Trp41 needed to establish a continuous hydrogen bonding network poses an additional energetic bottleneck for proton conduction.

Facile chemical synthesis of l-4CN-Trp and incorporation into a pHLIP peptide enabled FRET study on peptide–membrane interactions.

The amino acids tryptophan and 4-cyanotryptophan constitute a dual FRET and PET pair, useful for various biological applications.

Fluorescent amino acids (FAAs) offer significant advantages over fluorescent proteins in applications where the fluorophore size needs to be limited or minimized. A long-sought goal in biological spectroscopy/microcopy is to...

The ester carbonyl stretching vibration has recently been shown to be a sensitive and convenient infrared (IR) probe of protein electrostatics due to the linear dependence of its frequency on local electric field. While an ester moiety can be easily incorporated into peptides via solid-phase synthesis, currently there is no method available to site-specifically incorporate it into a large protein. Herein, we show that it is possible to use a cysteine alkylation reaction to achieve this goal and demonstrate the feasibility of this simple method by successfully incorporating a methyl ester group (CH COOCH ) into a model peptide (YGGCGG), two amyloid-forming peptides derived from the insulin B chain and Aβ, and bovine serum albumin (BSA). IR results obtained with those peptide and protein systems further confirm the utility of this vibrational probe in monitoring, for example, the structural integrity of amyloid fibrils and ligand binding-induced changes in protein local hydration status.

Unnatural nucleosides possessing unique spectroscopic properties that mimic natural nucleobases in both size and chemical structure are ideally suited for spectroscopic measurements of DNA/RNA structure and dynamics in a site-specific manner. However, such unnatural nucleosides are scarce, which prompts us to explore the utility of a recently found unnatural nucleoside, 4-cyanoindole-2′-deoxyribonucleoside (4CNI-NS), as a site-specific spectroscopic probe of DNA. A recent study revealed that 4CNI-NS is a universal nucleobase that maintains the high fluorescence quantum yield of 4-cyanoindole and that among the four natural nucleobases, only guanine can significantly quench its fluorescence. Herein, we further show that the C≡N stretching frequency of 4CNI-NS is sensitive to the local environment, making it a useful site-specific infrared probe of oligonucleotides. In addition, we demonstrate that the fluorescence-quencher pair formed by 4CNI-NS and guanine can be used to quantitatively assess the binding affinity of a single-stranded DNA to the protein system of interest via fluorescence spectroscopy, among other applications. We believe that this fluorescence binding assay is especially useful as its potentiality allows high-throughput screening of DNA–protein interactions.

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