<i>Cis</i>−<i>Trans</i>Photoisomerization of Fluorescent-Protein Chromophores

Voliani, Valerio; Bizzarri, Ranieri; Nifosı̀, Riccardo; Abbruzzetti, Stefania; Grandi, Elena; Viappiani, Cristiano; Beltram, Fabio

doi:10.1021/jp802419h

Cited by 116 publications

(183 citation statements)

References 37 publications

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“…Absorption of neutral and cationic p-HBDI peaks at 368 and 393 nm respectively, and displays a much narrower solvatochromism (variation of~20 nm, around 1,000 cm À1 ). Besides the main band, reported in Table 2, the absorption spectra of both neutral and anionic p-HBDI reveal features at shorter wavelength (around 300 and 250 nm for p-HBDI and at 320 nm for p-HBDI) [55,57]. Similar blueshifted bands are also measured in AHBMI (model chromophore of asFP and KFP) [50] and in model chromophores of BFPF, BFP, [55], and Kaede [60].…”

Section: Solutionsupporting

confidence: 70%

“…Besides the main band, reported in Table 2, the absorption spectra of both neutral and anionic p-HBDI reveal features at shorter wavelength (around 300 and 250 nm for p-HBDI and at 320 nm for p-HBDI) [55,57]. Similar blueshifted bands are also measured in AHBMI (model chromophore of asFP and KFP) [50] and in model chromophores of BFPF, BFP, [55], and Kaede [60]. Features around 330 nm are also detectable in red FPs [67].…”

Section: Solutionmentioning

confidence: 86%

“…Varying the solution pH, anionic, neutral, and cationic forms of p-HBDI are obtained, with pKa~8 for the anionic to neutral reaction and pKa~2 for the neutral to cationic reaction [17,57]. Analogous blue fluorescent protein (BFP, with the Y66H mutation), BFPF (BFP, with Y66F), and cyan fluorescent protein (CFP, with Y66W) chromophore models were synthesized [55]. Attempts to synthesize models for the RFP chromophore revealed that the compound with the acylimine group is not stable in solution, due to the susceptibility of acylimines to nucleophilic attack.…”

Section: Isolated Chromophoresmentioning

confidence: 99%

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One-Photon and Two-Photon Excitation of Fluorescent Proteins

Nifosı̀

Tozzini

2011

Springer Series on Fluorescence

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Fluorescent proteins (FPs) offer a wide palette of colors for imaging applications. One purpose of this chapter is to review the variety of FP spectral properties, with a focus on their structural basis. Fluorescence in FPs originates from the autocatalytically formed chromophore. Several studies exist on synthetic chromophore analogs in gas phase and in solution. Together with the X-ray structures of many FPs, these studies help to understand how excitation and emission energies are tuned by chromophore structure, protonation state, and interactions with the surrounding environment, either solvent molecules or amino acids residues. The increasing use of FPs in two-photon microscopy also prompted detailed investigations of their two-photon excitation properties. The comparison with one-photon excitation reveals nontrivial features, which are relevant both for their implications in understanding multiphoton properties of fluorophores and for application purposes.

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Section: Solutionsupporting

confidence: 70%

Section: Solutionmentioning

confidence: 86%

Section: Isolated Chromophoresmentioning

confidence: 99%

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One-Photon and Two-Photon Excitation of Fluorescent Proteins

Nifosı̀

Tozzini

2011

Springer Series on Fluorescence

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“…5. Cis/trans isomerization is a well-documented phenomenon, and is one of the reasons that the isolated analogues of fluorescent protein chromophores are nonfluorescent in solution [91]. In the isolated chromophore the cis/trans isomerization suppresses the fluorescence emission by offering a competitive pathway for deactivation of the electronically excited state.…”

Section: Natural Photoswitchesmentioning

confidence: 99%

Photoswitches: Key molecules for subdiffraction‐resolution fluorescence imaging and molecular quantification

Heilemann

Dedecker

Hofkens

et al. 2009

Laser & Photonics Reviews

249

191

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Optical microscopes, often referred to as 'light microscopes', use visible light and a system of lenses to provide us with magnified images of small samples. Combined with highly sensitive fluorescence detection techniques and efficient fluorescent probes they allow the non-invasive 3D study of subcellular structures even in living cells or tissue. However, optical microscopes are subject to the diffraction barrier of light which imposes an optical resolution limit of approximately 200 nm in the imaging plane. In the recent past new techniques emerged that break the diffraction barrier and enable structural investigations with so far unmatched resolution. They are all based on the selective switching of fluorophores between a fluorescent and a nonfluorescent state and are therefore generalized under the denotation "Photoswitching Microscopy". Here we review recent progress in subdiffraction-resolution fluorescence imaging microscopy using various photoswitchable fluorophores and strategies. Special emphasis will be placed on the design and development of photoswitches and the requirements photoswitches have to fulfill for successful use in photoswitching microscopy. Moreover, we demonstrate how photoswitches can be used advantageously for molecular quantification, i.e. the determination of densities and absolute numbers of proteins located in specific subcellular compartments and discuss concepts how standard organic fluorophores can be used successfully for photoswitching microscopy.All subdiffraction-resolution fluorescence imaging methods are based on the separation of fluorescence emission in time. Thus, they critically depend on the availability of photoswitches, i.e. molecules that can be switched between a fluorescent and nonfluorescent state upon irradiation with light with high reliability. Here we describe the development and operation methods of diverse photoswitches and their application for superresolution fluorescence imaging and molecular quantification.

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“…Photochromic/photoswitchable FPs are under continuous development, and the various mechanisms/factors that govern photoswitching have recently been reviewed [155,168,244]. Switching is thought to occur primarily through a cis-trans isomerization of the FP chromophore, as demonstrated with isolated synthetic chromophore analogues [245,246], however the chromophore environment within the FP structure also plays a key role in determining a FPs switchability [78]. Recently, it has been discovered that substitution of certain key amino acids in the chromophore environment within the FP structure can improve and/or restore the photochromic behavior of the FP chromophore, leading to improved photoswitchable FP mutations for pcFRET studies [78, 244,247].…”

Section: Green Fluorescent Protein and Derivativesmentioning

confidence: 99%

Materials for FRET Analysis: Beyond Traditional Dye–Dye Combinations

Sapsford

Wildt

Mariani

et al. 2013

FRET – Förster Resonance Energy Transfer

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The intrinsic sensitivity (r 6 dependence) of F€ orster (or fluorescence) resonance energy transfer (FRET) to nanoscale changes in the donor/acceptor separation distance has made FRET an invaluable biophysical tool in a variety of applications, ranging from studying the structure and conformation of proteins and nucleic acids to examining biomolecular interactions, including its use in in vitro and in vivo bioassays [1][2][3][4][5][6][7]. While the myriad of FRET configurations and techniques currently in use are covered throughout this book, here we focus primarily on the materials utilized as donor or acceptor probes in FRET rather than the process itself [3,5,8,9]. Our 2006 review paper on this topic serves as the foundation for this updated chapter [3]. The materials were divided into three main categories: organic materials that include "traditional" dye fluorophores, dark quenchers, polymers, and carbon nanomaterials (NMs); inorganic materials such as metal chelates, metal, and semiconductor nanocrystals; and fluorophores of biological origin such as fluorescent proteins (FPs), amino acids, and fluorescence generated from enzymatic bioluminescence (BL) and chemiluminescence (CL). These materials may function as FRET donors and/or acceptors, depending upon experimental design. Many of the new materials developed and/or new donor-acceptor probe combinations used address some of the inherent complications of more traditional FRET materials, including photobleaching, spectral cross talk, and direct excitation of the acceptor species, and examples of these will be highlighted and discussed throughout the chapter. Since the vast majority of FRET applications are biological in nature, they routinely involve some type of biomolecule labeling strategy, which ultimately plays a significant and fundamental role in the success and interpretation of the resulting FRET. Therefore, we begin the chapter with a brief discussion of the bioconjugation techniques commonly utilized for FRET and points to consider, followed by sections highlighting the current and emerging materials that are used, or have the potential to be used, in FRET applications.

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Cis−TransPhotoisomerization of Fluorescent-Protein Chromophores

Cited by 116 publications

References 37 publications

One-Photon and Two-Photon Excitation of Fluorescent Proteins

One-Photon and Two-Photon Excitation of Fluorescent Proteins

Photoswitches: Key molecules for subdiffraction‐resolution fluorescence imaging and molecular quantification

Materials for FRET Analysis: Beyond Traditional Dye–Dye Combinations

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