4-Cyanoindole-based fluorophores for biological spectroscopy and microscopy

Acharyya, Arusha; Ahmed, Ismail A.; Gai, Feng

doi:10.1016/bs.mie.2020.04.014

Cited by 7 publications

(5 citation statements)

References 57 publications

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“…Labeled peptides were then cleaved from dried resin and purified by preparative scale RP-HPLC, and their masses analyzed using MALDI and electrospray ionization mass spectrometry. Reprinted with permission from ref . Copyright 2020 Elsevier.…”

Section: Methodsmentioning

confidence: 99%

Visualization of Platelet Integrins via Two-Photon Microscopy Using Anti-transmembrane Domain Peptides Containing a Blue Fluorescent Amino Acid

et al. 2021

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The fluorescent reporters commonly used to visualize proteins can perturb both protein structure and function. Recently, we found that 4-cyanotryptophan (4CN-Trp), a blue fluorescent amino acid, is suitable for one-photon imaging applications. Here, we demonstrate its utility in two-photon fluorescence microscopy by using it to image integrins on cell surfaces. Specifically, we used solid-phase peptide synthesis to generate CHAMP peptides labeled with 4-cyanoindole (4CNI) at their N-termini to image integrins on cell surfaces. CHAMP (computed helical anti-membrane protein) peptides spontaneously insert into membrane bilayers to target integrin transmembrane domains and cause integrin activation. We found that 4CNI labeling did not perturb the ability of CHAMP peptides to insert into membranes, bind to integrins, or cause integrin activation. We then used two-photon fluorescence microscopy to image 4CNI-containing integrins on the surface of platelets. Compared to a 4CNI-labeled scrambled peptide that uniformly decorated cell surfaces, 4CNI-labeled CHAMP peptides were present in discrete blue foci. To confirm that these foci represented CN peptide-containing integrins, we co-stained platelets with integrin-specific fluorescent monoclonal antibodies and found that CN peptide and antibody fluorescence coincided. Because 4CNI can readily be biosynthetically incorporated into proteins with little if any effect on protein structure and function, it provides a facile way to directly monitor protein behavior and protein–protein interactions in cellular environments. In addition, these results clearly demonstrate that the two-photon excitation cross section of 4CN-Trp is sufficiently large to make it a useful two-photon fluorescence reporter for biological applications.

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Section: Methodsmentioning

confidence: 99%

Visualization of Platelet Integrins via Two-Photon Microscopy Using Anti-transmembrane Domain Peptides Containing a Blue Fluorescent Amino Acid

et al. 2021

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“…13.7 ns), and (3) it is chemically and photophysically stable (Figure ). Taken together, these spectroscopic attributes make 4CN-Trp a novel blue fluorescence UAA that is useful in both biological spectroscopy and microscopy. − For example, Hilaire et al used it to image the interaction of an antimicrobial peptide, Mastoparan X (MpX), with HEK293 T /17 cells via confocal microscopy (Figure ); and Acharyya et al demonstrated that the brightness of 4CN-Trp is large enough to allow visualization of the cellular localization of a peptide derived from the REV peptide of HIV through the use of a commercial fluorescence microscopy (Figure ). Similarly, Bennett and co-workers showed that 4CN-Trp containing molecules can be imaged via two-photon fluorescence microscopy and utilized it to image 4CNI-containing integrins on the surface of platelets (Figure ).…”

Section: Fluorescence Probesmentioning

confidence: 99%

“…Then the cells were fixed and imaged using a Leica DM6000 widefield fluorescence microscope equipped with a standard DAPI filter set. Reproduced with permission from ref . Copyright 2020 Elsevier B.V.…”

Section: Fluorescence Probesmentioning

confidence: 99%

Unnatural Amino Acids for Biological Spectroscopy and Microscopy

Feng,

Wang,

Zhang

et al. 2024

Chem. Rev.

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Due to advances in methods for site-specific incorporation of unnatural amino acids (UAAs) into proteins, a large number of UAAs with tailored chemical and/or physical properties have been developed and used in a wide array of biological applications. In particular, UAAs with specific spectroscopic characteristics can be used as external reporters to produce additional signals, hence increasing the information content obtainable in protein spectroscopic and/or imaging measurements. In this Review, we summarize the progress in the past two decades in the development of such UAAs and their applications in biological spectroscopy and microscopy, with a focus on UAAs that can be used as site-specific vibrational, fluorescence, electron paramagnetic resonance (EPR), or nuclear magnetic resonance (NMR) probes. Wherever applicable, we also discuss future directions.

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“…Fluorescence images of HeLa cells treated with a 4CN-indole labeled Rev peptide of 5 μM (A) and 10 mM (B) concentrations, collected on a Leica DM6000 widefield fluorescence microscope. Reprinted with permission from ref . Copyright 2020 Elsevier.…”

Section: Emerging Visible Fluorescent Amino Acidsmentioning

confidence: 99%

Tryptophan as a Template for Development of Visible Fluorescent Amino Acids

2021

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Most biological systems, at both molecular and cellular levels, are intrinsically complex, diverse, and nonfluorescent. Therefore, studying their structures, dynamics, and interactions via fluorescence-based methods requires incorporation of one or multiple external fluorophores that would not significantly affect any native property of the system in question. This requirement necessitates the development of a diverse set of fluorescence reporters that differ in chemical, physical, and photophysical properties. Herein, we offer our perspective on the need for, recent progress in, and future directions of developing tryptophan-based fluorescent amino acids.

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4-Cyanoindole-based fluorophores for biological spectroscopy and microscopy

Cited by 7 publications

References 57 publications

Visualization of Platelet Integrins via Two-Photon Microscopy Using Anti-transmembrane Domain Peptides Containing a Blue Fluorescent Amino Acid

Visualization of Platelet Integrins via Two-Photon Microscopy Using Anti-transmembrane Domain Peptides Containing a Blue Fluorescent Amino Acid

Unnatural Amino Acids for Biological Spectroscopy and Microscopy

Tryptophan as a Template for Development of Visible Fluorescent Amino Acids

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