Effects of reverse genetic mutations on the spectral and photochemical behavior of a photoactivatable fluorescent protein PAiRFP1 (original) (raw)
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Biomolecules, 2020
Two photoactivatable near infrared fluorescent proteins (NIR FPs) named "PAiRFP1" and "PAiRFP2" are formed by directed molecular evolution from Agp2, a bathy bacteriophytochrome of Agrobacterium tumefaciens C58. There are 15 and 24 amino acid substitutions in the structure of PAiRFP1 and PAiRFP2, respectively. A comprehensive molecular exploration of these bacteriophytochrome photoreceptors (BphPs) are required to understand the structure dynamics. In this study, the NIR fluorescence emission spectra for PAiRFP1 were recorded upon repeated excitation and the fluorescence intensity of PAiRFP1 tends to increase as the irradiation time was prolonged. We also predicted that mutations Q168L, V244F, and A480V in Agp2 will enhance the molecular stability and flexibility. During molecular dynamics (MD) simulations, the average root mean square deviations of Agp2, PAiRFP1, and PAiRFP2 were found to be 0.40, 0.49, and 0.48 nm, respectively. The structure of PAiRFP1 and PAi...
On the Origin of Fluorescence in Bacteriophytochrome Infrared Fluorescent Proteins
The Journal of …, 2010
Tsien (Science, 2009, 324, 804-807) has recently reported the creation of the first infrared fluorescent protein (IFP). It was engineered from bacterial phytochrome by removing the PHY and histidine kinase-related domains, by optimizing the protein to prevent dimerization and by limiting the biliverdins conformational freedom, especially around its D ring. We have used database analyses and molecular dynamics simulations with freely rotating chromophoric dihedrals in order to model the dihedral freedom available to the biliverdin D ring in the excited state; to show that the tetrapyrrole ligands in phytochromes are flexible and can adopt many conformations, however their conformational space is limited/defined by the chemospatial characteristics of the protein cavity. Our simulations confirm that the reduced accessibility to conformations geared to an excited state proton transfer may be responsible for the fluorescence in IFP, just as has been suggested by Kennis (PNAS, 2010, 107, 9170-9175) for fluorescent bacteriophytochrome from Rhodopseudomonas palustris.
Nature Communications, 2013
The ability to modulate the fluorescence of optical probes can be used to enhance signal-tonoise ratios for imaging within highly autofluorescent environments, such as intact tissues and living organisms. Here we report two bacteriophytochrome-based photoactivatable nearinfrared fluorescent proteins, named PAiRFP1 and PAiRFP2. PAiRFPs utilize haem-derived biliverdin, ubiquitous in mammalian tissues, as the chromophore. Initially weakly fluorescent PAiRFPs undergo photoconversion into a highly fluorescent state with excitation/emission at 690/717 nm following a brief irradiation with far-red light. After photoactivation, PAiRFPs slowly revert back to initial state, enabling multiple photoactivation-relaxation cycles. Low-temperature optical spectroscopy reveals several intermediates involved in PAiRFP photocycles, which all differ from that of the bacteriophytochrome precursor. PAiRFPs can be photoactivated in a spatially selective manner in mouse tissues, and optical modulation of their fluorescence allows for substantial contrast enhancement, making PAiRFPs advantageous over permanently fluorescent probes for in vivo imaging conditions of high autofluorescence and low signal levels.
Biophysical Journal - BIOPHYS J, 2010
Phytochromes are red-light photoreceptor proteins that regulate a variety of responses and cellular processes in plants, bacteria, and fungi. The phytochrome light activation mechanism involves isomerization around the C15═C16 double bond of an open-chain tetrapyrrole chromophore, resulting in a flip of its D-ring. In an important new development, bacteriophytochrome (Bph) has been engineered for use as a fluorescent marker in mammalian tissues. Here we report that an unusual Bph, RpBphP3 from Rhodopseudomonas palustris, denoted P3, is fluorescent. This Bph modulates synthesis of light-harvesting complex in combination with a second Bph exhibiting classical photochemistry, RpBphP2, denoted P2. We identify the factors that determine the fluorescence and isomerization quantum yields through the application of ultrafast spectroscopy to wild-type and mutants of P2 and P3. The excited-state lifetime of the biliverdin chromophore in P3 was significantly longer at 330-500 ps than in P2 and other classical phytochromes and accompanied by a significantly reduced isomerization quantum yield. H/D exchange reduces the rate of decay from the excited state of biliverdin by a factor of 1.4 and increases the isomerization quantum yield. Comparison of the properties of the P2 and P3 variants shows that the quantum yields of fluorescence and isomerization are determined by excited-state deprotonation of biliverdin at the pyrrole rings, in competition with hydrogen-bond rupture between the D-ring and the apoprotein. This work provides a basis for structure-based conversion of Bph into an efficient near-IR fluorescent marker.
Photoinduced Chemistry in Fluorescent Proteins: Curse or Blessing?
Chemical Reviews, 2017
Photoinduced reactions play an important role in the photocycle of fluorescent proteins from the green fluorescent protein (GFP) family. Among such processes are photoisomerization, photooxidation/photoreduction, breaking and making of covalent bonds, and excited-state proton transfer (ESPT). Many of these transformations are initiated by electron transfer (ET). The quantum yields of these processes vary significantly, from nearly 1 for ESPT to 10 −4 −10 −6 for ET. Importantly, even when quantum yields are relatively small, at the conditions of repeated illumination the overall effect is significant. Depending on the task at hand, fluorescent protein photochemistry is regarded either as an asset facilitating new applications or as a nuisance leading to the loss of optical output. The phenomena arising due to phototransformations include (i) large Stokes shifts, (ii) photoconversions, photoactivation, and photoswitching, (iii) phototoxicity, (iv) blinking, (v) permanent bleaching, and (vi) formation of long-lived intermediates. The focus of this review is on the most recent experimental and theoretical work on photoinduced transformations in fluorescent proteins. We also provide an overview of the photophysics of fluorescent proteins, highlighting the interplay between photochemistry and other channels (fluorescence, radiationless relaxation, and intersystem crossing). The similarities and differences with photochemical processes in other biological systems and in dyes are also discussed.
2002
Mutants of green fluorescent protein (GFP) are usually designed to absorb and emit light as ''one color'' systems, i.e. with a single, photostable conformation of the chromophore. We have studied two red-shifted GFP-mutants (S65T and EYFP) by means of hole-burning and high-resolution optical spectroscopy at low temperature, and compare the data to those previously reported for RS-GFP. We prove that these GFP-mutants are not ''one color'' systems because they can be reversibly phototransformed from one conformation into another. The results are rationalized in terms of energylevel schemes that are similar to that previously derived by us for wild-type GFP. In these schemes, each mutant can be interconverted by light among at least three conformations that are associated with the protonation-state of the chromophore. The results have relevance for the study of protein-protein interactions by fluorescence resonance energy transfer (FRET), where GFP-mutants of different colors are used as labels in donor-acceptor pairs. Furthermore, we present a detailed mechanism that explains the ''on-off'' and ''blinking'' behavior of single GFP-molecules with the proposed energy-level diagrams.
Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology, 2016
The genome of the cyanobacterium Nostoc sp. PCC 7120 encodes a large number of putative bacteriophytochrome and cyanobacteriochrome photoreceptors that, due to their long-wavelength absorption and fluorescence emission, might serve as fluorescent tags in intracellular investigations. We show that the PAS-GAF domain of the bacteriophytochrome, AphB, binds biliverdin covalently and exhibits, besides its reversible photochemistry, a moderate fluorescence in the near infrared (NIR) spectral region. It was selected for further increasing the brightness while retaining the NIR fluorescence. In the first step, amino acids assumed to improve fluorescence were selectively mutated. The resulting variants were then subjected to several rounds of random mutagenesis and screened for enhanced fluorescence in the NIR. The brightness of optimized PAS-GAF variants increased more than threefold compared to that of wt AphB(1-321), with only insignificant spectral shifts (Amax around 695 nm, and Fmax a...
On excited state reaction path in reversibly switchable fluorescent proteins
Cornell University - arXiv, 2017
A set formed by five reversibly-switchable fluorescent proteins (RSFPs) display spread over 40 nm in absorption maxima and only 18 nm in emission. The five proteins-Dronpa, rsFastLime, rsKame, Padron(anionic form) and bsDronpa-carry exactly the same chromophore and differ just in a few mutations. Thus they form an ideal set for mechanistic investigation. Starting with the results of molecular dynamics simulations we use QM/MM calculations to investigate the effects controlling the spectral tuning. In this contribution we show that the models, which are based on CASPT2//CASSCF level of QM theory, reproduce the observed absorption trend with only a limited blue-shift of 4.5 kcal/mol and emission trend with even smaller blue-shift of 1.5 kcal/mol. Using CASSCF QM/MM calculations we analyze the chromophore's charge-transfer patterns during the absorption and emission, which, in turn trigger a cascade of hydrogen-bond-network rearrangements indicating a preparation to an isomerization event. We also show how the contribution of individual aminoacids to the chromophore conformational changes correlates with spectral tuning of the absorption and emission. Furthermore, we identify how the conical intersection topography correlates with protein's photophysical properties. In conclusion, we establish a detailed mechanistic explanation of variations in photo-switching speed as well as higher sensitivity of RSFPs to mutation observed for light absorption relative to light emission.
Protein-chromophore interactions in photoreceptors often shift the chromophore absorbance maximum to a biologically relevant spectral region. A fundamental question regarding such spectral tuning effects is how the electronic ground state S 0 and excited state S 1 are modified by the protein. It is widely assumed that changes in energy gap between S 0 and S 1 are the main factor in biological spectral tuning. We report a generally applicable approach to determine if a specific residue modulates the energy gap, or if it alters the equilibrium nuclear geometry or width of the energy surfaces. This approach uses the effects that changes in these three parameters have on the absorbance and fluorescence emission spectra of mutants. We apply this strategy to a set of mutants of photoactive yellow protein (PYP) containing all 20 side chains at active site residue 46. While the mutants exhibit significant variation in both the position and width of their absorbance spectra, the fluorescence emission spectra are largely unchanged. This provides strong evidence against a major role for changes in energy gap in the spectral tuning of these mutants and reveals a change in the width of the S 1 energy surface. We determined the excited state lifetime of selected mutants and the observed correlation between the fluorescence quantum yield and lifetime shows that the fluorescence spectra are representative of the energy surfaces of the mutants. These results reveal that residue 46 tunes the absorbance spectrum of PYP largely by modulating the width of the S 1 energy surface. photoreceptor | protein-chromophore interactions | wavelength regulation L ight-driven proteins, consisting of a protein-chromophore complex, employ two general strategies to optimize the biological effectiveness of the position of their absorbance spectra. First, covalent modifications of a chromophore can shift its absorbance maximum λ abs max. An important example is the strong red-shift in the absorbance spectrum of bacteriochlorophyll compared to plant chlorophyll (1). Second, specific protein-chromo-phore interactions can shift the absorbance spectrum of the protein-bound chromophore. A classic example of this spectral tuning phenomenon is color vision in vertebrates (2, 3): the same retinal chromophore can absorb in the blue, green, and red, depending on the amino acid sequence of the rhodopsin to which it is bound. Spectral tuning provides an example of protein-ligand interactions that tune the properties of the cofactor to biologically relevant values. Two main approaches have been used to unravel the factors involved in spectral tuning: determining (i) which amino acids in the protein contribute to spectral tuning, and (ii) what type of protein-chromophore interactions cause spectral tuning. Interactions that alter the degree of charge delocalization over the chromophore are of particular importance. These two issues have been explored extensively in animal visual rhodopsins and the ar-chaeal rhodopsins (4-7). Here we study spectral tuning in a more recently discovered photoreceptor, photoactive yellow protein (PYP) (8, 9), and use it to develop and explore a third approach to investigate spectral tuning. Central in this approach are the effects of an individual residue on the shape of the S 0 ground state and S 1 excited state energy surfaces. PYP offers a highly accessible system (10) to study spectral tuning. We use it here to test a widely used assumption that has dominated the field of biological spectral tuning: that changes in the energy gap ΔE between the S 0 and S 1 energy surfaces are the major factor in spectral tuning. PYP is a blue light receptor from the halophilic photosynthetic eubacterium Halorhodospira halophila (8, 9). It functions as the photoreceptor for negative phototaxis in H. halophila (11). The purified protein exhibits a photocycle (9, 12) initiated by the photoisomerization (13) of its p-coumaric acid (pCA) chromo-phore (14, 15) by a flip in the position of its carbonyl group (16). PYP consists of an antiparallel 6-stranded β-sheet flanked by five α-helices (17). Its pCA chromophore is fully buried within the protein and its deprotonated phenolic oxygen forms short hydrogen bonds with the side chains of Tyr42 and Glu46 (Fig. 1A). Site-directed mutagenesis studies (summarized in ref. 18) have identified Glu46 as a key factor in the spectral tuning of PYP (19-21). Three major factors contribute to spectral tuning in PYP (22) (Fig. 1B). First, the formation of the thioester bond between the pCA and Cys69 results in a red-shift from 284 to 335 nm. Second, the deprotonation of the phenolic oxygen of the pCA causes a further red-shift to 400 nm. The strong downshift in the pK a of the pCA from 8.8 in solution (22) to 2.8 in PYP (8, 23) ensures that the chromophore in PYP is deprotonated under physiological conditions. Finally, specific protein-chromophore interactions result in the final red-shift to 446 nm. These interactions have been studied by both site-directed mutagenesis (18-21) and computational approaches (24-27) (SI Text). These studies have revealed Tyr42 and Glu46 as the two dominant side chains in the spectral tuning of PYP (18-21, 24-28). Weakening of the Glu46-pCA hydrogen bond (29, 30) in the E46Q mutant results in a red-shift to 460 nm, while complete disruption of the hydrogen bond in the E46V mutant results in a further red-shift to 478 nm (21). Based on computational studies it has been proposed that hydrogen bonding, charge-charge interactions, and burial of the pCA in the hydrophobic protein interior are factors in the spectral tuning of PYP (24, 25). Here we propose a straightforward approach to probe if spectral tuning alters not only the ΔE but also the equilibrium nuclear geometry R e or the width W of the S 0 and S 1 energy surfaces. We