A naturally monomeric infrared fluorescent protein for protein labeling in vivo (original) (raw)
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2019
Bacteriophytochrome photoreceptors (BphPs) containing biliverdin (BV) have great potential for the development of genetically engineered near-infrared fluorescent proteins (NIR FPs). We investigated a photoactivatable fluorescent protein PAiRFP1, was engineered through directed molecular evolution. The coexistence of both red light absorbing (Pr) and far-red light absorbing (Pfr) states in dark is essential for the photoactivation of PAiRFP1. The PCR based site-directed reverse mutagenesis, spectroscopic measurements and molecular dynamics (MD) simulations were performed on three targeted sites V386A, V480A and Y498H in PHY domain to explore their potential effects during molecular evolution of PAiRFP1. We found that these substitutions did not affect the coexistence of Pr and Pfr states but led to slight changes in the photophysical parameters. The covalent docking of biliverdin (cis and trans form) with PAiRFP1 was followed by several 100 ns MD simulations to provide some theoretical explanations for the coexistence of Pr and pfr states. The results suggested that experimentally observed coexistence of Pr and Pfr states in both PAiRFP1 and mutants were resulted from the improved stability of Pr state. The use of experimental and computational work provided useful understanding of Pr and Pfr states and the effects of these mutations on the photophysical properties of PAiRFP1.
EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion
Proceedings of The National Academy of Sciences, 2004
A gene encoding a fluorescent protein from the stony coral Lobophyllia hemprichii has been cloned in Escherichia coli and characterized by biochemical and biophysical methods. The protein, which we named EosFP, emits strong green fluorescence (516 nm) that changes to red (581 nm) upon near-UV irradiation at Ϸ390 nm because of a photo-induced modification involving a break in the peptide backbone next to the chromophore. Single-molecule fluorescence spectroscopy shows that the wild type of EosFP is tetrameric, with strong Fö rster resonance coupling among the individual fluorophores. We succeeded in breaking up the tetramer into AB and AC subunit dimers by introducing the single point mutations V123T and T158H, respectively, and the combination of both mutations yielded functional monomers. Fusion constructs with a variety of proteins were prepared and expressed in human cells, showing that normal biological functions were retained. The possibility to locally change the emission wavelength by focused UV light makes EosFP a superb marker for experiments aimed at tracking the movements of biomolecules within the living cell.
Bright monomeric red fluorescent protein with an extended fluorescence lifetime
Nature Methods, 2007
Fluorescent proteins have become extremely popular tools for in vivo imaging and especially for the study of localization, motility and interaction of proteins in living cells. Here we report TagRFP, a monomeric red fluorescent protein, which is characterized by high brightness, complete chromophore maturation, prolonged fluorescence lifetime and high pH-stability. These properties make TagRFP an excellent tag for protein localization studies and fluorescence resonance energy transfer (FRET) applications.
The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins
Trends in biochemical sciences, 2016
Over the past 20 years, protein engineering has been extensively used to improve and modify the fundamental properties of fluorescent proteins (FPs) with the goal of adapting them for a fantastic range of applications. FPs have been modified by a combination of rational design, structure-based mutagenesis, and countless cycles of directed evolution (gene diversification followed by selection of clones with desired properties) that have collectively pushed the properties to photophysical and biochemical extremes. In this review, we provide both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems.
Scientific Reports, 2013
Most GFP-like fluorescent proteins exhibit small Stokes shifts (10-45 nm) due to rigidity of the chromophore environment that excludes non-fluorescent relaxation to a ground state. An unusual near-infrared derivative of the red fluorescent protein mKate, named TagRFP675, exhibits the Stokes shift, which is 30 nm extended comparing to that of the parental protein. In physiological conditions, TagRFP675 absorbs at 598 nm and emits at 675 nm that makes it the most red-shifted protein of the GFP-like protein family. In addition, its emission maximum strongly depends on the excitation wavelength. Structures of TagRFP675 revealed the common DsRed-like chromophore, which, however, interacts with the protein matrix via an extensive network of hydrogen bonds capable of large flexibility. Based on the spectroscopic, biochemical, and structural analysis we suggest that the rearrangement of the hydrogen bond interactions between the chromophore and the protein matrix is responsible for the TagRFP675 spectral properties. G FP-like fluorescent proteins (FPs) are indispensable imaging tools for all areas of biomedical research 1-3. Three dimensional structures of GFP-like proteins are highly conserved, consisting of a beta-barrel formed by about 220-240 amino acids. A chromophore is buried inside the barrel, shielded by the protein matrix from stochastic interactions with solvent molecules. The rigid environment provided by protein scaffold prevents thermal isomerization and nonfluorescent relaxation of the chromophore. A wide variety of FP spectral phenotypes originate from two major contributing factors: the chemical structure of the chromophore, and interactions occurring between the chromophore, both in the ground and excited states, and its immediate environment. Based on the chemical structure, which to a large extent determines the spectral properties of FPs, the chromophores can be classified into several groups 4. Most of the red and far-red FPs contain so called DsRedlike chromophores 5 , which can exist in either neutral or anionic states. Neutral DsRed-like chromophores absorb blue-cyan light and emit green-yellow, whereas the anionic forms possess excitation and emission maxima at ,560-580 and 570-610 nm, respectively 4,6,7. However, fluorescence spectra can be significantly perturbed by changes in the immediate chromophore environment. Spectroscopic studies combined with high resolution crystal structures revealed the interactions responsible for the bathochromic shift of fluorescence in several red and far-red FPs. Among the most common modifications of the chromophore environment in far-red FPs is the introduction of a hydrogen bond between the chromophore and its immediate environment. An important example is the hydrogen bond between the N-acylimine oxygen of the DsRed-like chromophore and a water molecule or a side chain of an amino acid (Figure 1A). This type of interaction has been observed in mNeptune 8 , eqFP650 (Ref. 9,10), eqFP670 (Ref. 9,10), mRojoA 11 , mRouge 11 , which possess water-mediated hydrogen bonds. In mPlum 12 and its variant mPlum/E16Q 13 , the N-acylimine oxygen of the chromophore forms direct hydrogen bonds with the side chain functionalities of Glu16 and Gln16, respectively 13. A hydrogen bond between the protonated Glu215 carboxyl group and the imidazolinone ring nitrogen was proposed to account for the