The Rainbow Arching over the Fluorescent Thienoviologen Mesophases (original) (raw)
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Biliproteins are photosynthetic light-harvesting proteins, which transfer excitons with high efficiencies over relatively long distances until they arrive at a photosynthetic reaction center. Purified R-phycoerythrin (isolated from a red alga) and C-phycoerythrin (isolated from a cyanobacterium), each of which contains several chromophores, were studied by a combination of fluorescence emission, fluorescence excitation polarization, and absorption methods. The polarization spectra of both these biliproteins showed that there was a minimum of two spectrally distinct sensitizing chromophores, which, after absorbing photons, transfer excitons to the lowest-energy (fluorescing) chromophores. Some of these spectroscopic data were used to deconvolute the absorption spectra into the spectra of the two sensitizing and one fluorescing chromophores. It was shown thai the higher-energy sensitizing chromophore could readily transfer its excitation energy to the fluorescing chromophore using the lower-energy sensitizing chromophore as an intermediary. However, there was sufficient spectral overlap between the higher-energy sensitizing chromophore and the fluorescing chromophore so that direct transfer between them could not be ruled out.
Spectral and structural comparison between bright and dim green fluorescent proteins in Amphioxus
Scientific Reports, 2014
The cephalochordate Amphioxus naturally co-expresses fluorescent proteins (FPs) with different brightness, which thus offers the rare opportunity to identify FP molecular feature/s that are associated with greater/lower intensity of fluorescence. Here, we describe the spectral and structural characteristics of green FP (bfloGFPa1) with perfect (100%) quantum efficiency yielding to unprecedentedly-high brightness, and compare them to those of co-expressed bfloGFPc1 showing extremely-dim brightness due to low (0.1%) quantum efficiency. This direct comparison of structure-function relationship indicated that in the bright bfloGFPa1, a Tyrosine (Tyr159) promotes a ring flipping of a Tryptophan (Trp157) that in turn allows a cis-trans transformation of a Proline (Pro55). Consequently, the FP chromophore is pushed up, which comes with a slight tilt and increased stability. FPs are continuously engineered for improved biochemical and/or photonic properties, and this study provides new insight to the challenge of establishing a clear mechanistic understanding between chromophore structural environment and brightness level. F irst discovered in 1961 by Shimomura and colleagues in the cnidarian jellyfish Aequorea victorea 1 , the green fluorescent protein (GFP) and its variants have become the cornerstone of fluorescent protein technologies, exponentially expanding the application of fluorescence spectroscopy and imaging in molecular, cellular and developmental biology, as well as the applied fields of biotechnology and bioengineering 2,3. Critical steps in the widespread use of GFP included the elucidation of the three dimensional structure of the wild-type GFP 4 and the S65T-GFP mutant that resulted in increased fluorescence, photostability and a red shifting the major excitation peak to 488 nm with the peak emission kept at 509 nm 5. These initial atomic resolution views of GFP highlighted the unusual architecture and key functional groups of this protein family described as 'paint in a can' 5. GFP folds into an 11-stranded b-barrel with a single helical segment threaded through the center of the barrel, and capped by several loops at either end of the barrel. A sharp turn places strain on three residues within the barrel-encapsulated stretch, which consequently drives cyclization via a dehydration/ oxidation mechanism that requires only molecular oxygen 6-8. The cyclized chromophore remains covalently attached to the polypeptide chain, and therefore, every cell expressing the GFP gene acquires fluorescence. This genetically encoded autonomy and self-assembly makes the GFP gene a powerful biological tag for the in vivo visualization of a wide array of cellular structures and processes 2,9,10. Since the publication of the landmark crystal structures from the Remington and Phillips groups, respectively 4,5 , more than 250 structures of engineered GFP variants from Aequorea victorea have been deposited into the Protein Data Bank (PDB), and are described in many publications (www.rcsb.org) 11. This extensive catalog of three-dimensional structures coupled with biochemical characterization has enabled both structure-guided engineering of the wild-type AvGFP and the ability to decipher the effect of random mutations and directed amino acid substitutions on the proteins' optical characteristics 11. These engineering efforts have produced GFPs with widely varying solubilities, oligomerization states, pH optima, halide and temperature sensitivities, and a diversity of fluorescence intensity, brightness, absorption and emission spectra (e.g. 12-14). While these engineering efforts produced impressive improvements in a broad array of biophysical parameters, they were mostly associated with production of green fluorescence, while the production of GFP-like proteins fluorescing in the orangered range of the visible spectrum remaining more challenging 15 .
Photophysical Behavior of mNeonGreen, an Evolutionarily Distant Green Fluorescent Protein
Biophysical journal, 2018
Fluorescent proteins (FPs) feature complex photophysical behavior that must be considered when studying the dynamics of fusion proteins in model systems and live cells. In this work, we characterize mNeonGreen (mNG), a recently introduced FP from the bilaterian Branchiostoma lanceolatum, in comparison to the well-known hydrozoan variants enhanced green fluorescent protein (EGFP) and Aequorea coerulescens GFP by steady-state spectroscopy and fluorescence correlation spectroscopy in solutions of different pH. Blind spectral unmixing of sets of absorption spectra reveals three interconverting electronic states of mNG: a nonfluorescent protonated state, a bright state showing bell-shaped pH dependence, and a similarly bright state dominating at high pH. The gradual population of the acidic form by external protonation is reflected by increased flickering at low pH in fluorescence correlation spectroscopy measurements, albeit with much slower flicker rates and lower amplitudes as compare...
Autofluorescence: Biological functions and technical applications
Plant Science, 2015
Chlorophylls are the most remarkable examples of fluorophores, and their fluorescence has been intensively studied as a non-invasive tool for assessment of photosynthesis. Many other fluorophores occur in plants, such as alkaloids, phenolic compounds and porphyrins. Fluorescence could be more than just a physicochemical curiosity in the plant kingdom, as several functional roles in biocommunication occur or have been proposed. Besides, fluorescence emitted by secondary metabolites can convert damaging blue and UV into wavelengths potentially useful for photosynthesis. Detection of the fluorescence of some secondary phytochemicals may be a cue for some pollinators and/or seed dispersal organisms. Independently of their functions, plant fluorophores provide researchers with a tool that allows the visualization of some metabolites in plants and cells, complementing and overcoming some of the limitations of the use of fluorescent proteins and dyes to probe plant physiology and biochemistry. Some fluorophores are influenced by environmental interactions, allowing fluorescence to be also used as a specific stress indicator.
Naturally occurring fluorescence in frogs
Proceedings of the National Academy of Sciences of the United States of America, 2017
Fluorescence, the absorption of short-wavelength electromagnetic radiation reemitted at longer wavelengths, has been suggested to play several biological roles in metazoans. This phenomenon is uncommon in tetrapods, being restricted mostly to parrots and marine turtles. We report fluorescence in amphibians, in the tree frog Hypsiboas punctatus, showing that fluorescence in living frogs is produced by a combination of lymph and glandular emission, with pigmentary cell filtering in the skin. The chemical origin of fluorescence was traced to a class of fluorescent compounds derived from dihydroisoquinolinone, here named hyloins. We show that fluorescence contributes 18-29% of the total emerging light under twilight and nocturnal scenarios, largely enhancing brightness of the individuals and matching the sensitivity of night vision in amphibians. These results introduce an unprecedented source of pigmentation in amphibians and highlight the potential relevance of fluorescence in visual ...
Green fluorescent proteins are light-induced electron donors
Nature Chemical Biology, 2009
Proteins of the green fluorescent protein (GFP) family are well known due to their unique biochemistry and extensive use as in vivo markers. Here, we discovered a new feature of GFPs of diverse origins to act as the light-induced electron donors in photochemical reactions with various electron acceptors, including biologically relevant ones. Moreover, this process accompanying with green-to-red GFP photoconversion can be observed in living cells without additional treatment. Green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its artificial mutants are widely used in cell biology and biomedical research as genetically encoded fluorescent markers1. In the past decade, a number of GFP-like proteins have been found in diverse marine creatures-corals2, copepods3, and even lower chordates4. Detailed biochemical and crystallographic studies have showed a remarkable diversity of chromophore structures that explain wide spectral variations in GFP-like proteins5-7. In addition, diverse photoconversions have been demonstrated for fluorescent proteins8,9. In particular, in 1997, an intriguing phenomenon was described: under anaerobic conditions GFP and some of its mutants underwent efficient photoconversion into a red fluorescent state10,11. We use the term "redding" to describe this photoconversion. Little is known about the structural basis of GFP redding. It occurs only if the oxygen concentration in the superfusing gas is below 1%12. Red fluorescence possesses excitation-emission maxima at 525 and 600 nm, respectively10, and a fluorescence lifetime of 2.5 ns13. The red fluorescent form is stable for many hours in the absence of molecular oxygen, but it disappears quickly after re-oxygenation of the sample10,12,13. The structure and mechanisms of the formation of the GFP red state are unclear, and no hypothesis has been proposed. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Reviewing the relevance of fluorescence in biological systems
Photochem. Photobiol. Sci., 2015
We review the state of the art in the research on the fluorescence emitted by plant leaves, fruits, flowers, avians, butterflies, beetles, dragonflies, millipedes, cockroaches, bees, spiders, scorpions and sea organisms and discuss its relevance in nature.