Time‐Resolved Fluorescence Study of the Dissociation of FMN from the Yellow Fluorescence Protein from <i>Vibrio fischeri</i>

Visser, Antonie J. W. G.; Hoek, A. van; Visser, Nina V.; Lee, Yongho; Ghisla, Sandro

doi:10.1111/j.1751-1097.1997.tb08607.x

Cited by 23 publications

(18 citation statements)

References 19 publications

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“…This observation is in full agreement with the three-dimensional structures in which the fluorophore is rigidly incorporated in the central helix (8 -10). The rigidity of the binding site seems a general property of fluorophores involved in bioluminescence; there is no internal motion of other light emitting antenna fluorophores as well (34,40). The hydrodynamic radius (R h ) calculated from the obtained rotational correlation time (Equation 4) is 2.21 nm (Table II) and in good agreement with the fluorescence correlation experiment.…”

Section: Resultsmentioning

confidence: 99%

“…Time-resolved polarized fluorescence experiments were carried out using a picosecond laser system and time-correlated single photon counting as described in detail elsewhere (32)(33)(34). The excitation wavelength was 480 nm (coumarin 150 dye as laser medium, pumped by a modelocked Nd-YLF laser), and the fluorescence was selected by using a bandpass filter (K50) in conjunction with a GG495 cut-off filter (both filters were from Schott, Mainz, Germany).…”

Section: ϫ2mentioning

confidence: 99%

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Structural Dynamics of Green Fluorescent Protein Alone and Fused with a Single Chain Fv Protein

Hink¹,

Griep²,

Borst³

et al. 2000

Journal of Biological Chemistry

185

132

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Structural information on intracellular fusions of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria with endogenous proteins is required as they are increasingly used in cell biology and biochemistry. We have investigated the dynamic properties of GFP alone and fused to a single chain antibody raised against lipopolysaccharide of the outer cell wall of Gram-negative bacteria (abbreviated as scFv-GFP). The scFv moiety was functional as was proven in binding assays, which involved the use of both fluorescence correlation spectroscopy observing the binding of scFv-GFP to Gram-negative bacteria and a surface plasmon resonance cell containing adsorbed lipopolysaccharide antigen. The rotational motion of scFv-GFP has been investigated with time-resolved fluorescence anisotropy. However, the rotational correlation time of scFv-GFP is too short to account for globular rotation of the whole protein. This result can only be explained by assuming a fast hinge motion between the two fused proteins. A modeled structure of scFv-GFP supports this observation.The green fluorescent protein (GFP) 1 from the jellyfish Aequorea victoria has received widespread utilization as a natural fluorescent marker for gene expression, localization of gene products (1-5), and identification of protein interaction and function. GFP is a protein consisting of 238 amino acids with a molecular mass of 27 kDa and has the shape of a cylinder with a length of 4.2 nm and diameter of 2.4 nm. The chemical structure of the hexapeptide chromophore has been elucidated (6). The intrinsic fluorophore is a p-hydroxybenzylidene-imidazolidine derivative formed by a covalent modification of the sequence Ser 65 (or Thr 65 in enhanced GFP), Tyr 66 , and Gly 67 in the hexapeptide. A comprehensive review on GFP has been published (7). The crystal structure of GFP and enhanced GFP has been solved and showed the hexapeptide to be part of a central helix inside a 11-stranded ␤-barrel (8 -11).Genetic fusions of a variety of proteins with GFP have been used in numerous studies on gene or protein function. In a sense it is miraculous that in most fusion proteins GFP is functional. In many other fusion proteins, the protein used as a reporter does often not fold well, resulting in aggregates or inclusion bodies of the entire fusion protein. Sometimes the causes of aggregation can be attributed to certain (clusters of) amino acids such as hydrophobic clusters of amino acids that become solvent exposed (12). To obtain a better picture why these phenomena do not occur in GFP fusion proteins, we have investigated the behavior of the GFP moiety in fusion proteins and emphasized the motional properties. Thereto, we fused enhanced GFP with a single chain Fv fragment raised against the lipopolysaccharide (LPS) of a Gram-negative bacterium. Because the single chain antibody was linked to the N-terminal residue of GFP, the fusion protein is abbreviated as scFv-GFP. Here we report information relevant for the dynamics of GFP fusion proteins used to monitor pro...

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Section: Resultsmentioning

confidence: 99%

Section: ϫ2mentioning

confidence: 99%

Structural Dynamics of Green Fluorescent Protein Alone and Fused with a Single Chain Fv Protein

Hink¹,

Griep²,

Borst³

et al. 2000

Journal of Biological Chemistry

185

132

View full text Add to dashboard Cite

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“…LumP and YFP are homologous (37%) (O'Kane et al, 1991) and also share homology with riboflavin synthase, which binds two molecules of the lumazine (O' Kane and Prasher, 1992). A remarkable property of YFP is that the bound FMN exhibits a long fluorescence lifetime (7.6 ns) and high quantum yield (0.6) commensurate with its function, as most flavoproteins are hardly fluorescent at all (Visser et al, 1997).…”

Section: Structure Of Lumazine Proteinmentioning

confidence: 99%

Protein-protein complexation in bioluminescence

et al. 2011

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In this review we summarize the progress made towards understanding the role of protein-protein interactions in the function of various bioluminescence systems of marine organisms, including bacteria, jellyfish and soft corals, with particular focus on methodology used to detect and characterize these interactions. In some bioluminescence systems, protein-protein interactions involve an "accessory protein" whereby a stored substrate is efficiently delivered to the bioluminescent enzyme luciferase. Other types of complexation mediate energy transfer to an "antenna protein" altering the color and quantum yield of a bioluminescence reaction. Spatial structures of the complexes reveal an important role of electrostatic forces in governing the corresponding weak interactions and define the nature of the interaction surfaces. The most reliable structural model is available for the protein-protein complex of the Ca 2+ -regulated photoprotein clytin and green-fluorescent protein (GFP) from the jellyfish Clytia gregaria, solved by means of Xray crystallography, NMR mapping and molecular docking. This provides an example of the potential strategies in studying the transient complexes involved in bioluminescence. It is emphasized that structural studies such as these can provide valuable insight into the detailed mechanism of bioluminescence.

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“…For the YPF's, LumYFP τ = 13.4 ns, RFYFP and FMNYFP τ = 7.6 ns (0°C). The natural YFP having FMN bound, has K D = 400 nM, τ = 7.6 ns and φ = 14.8 ns (20°C) .…”

Section: Introductionmentioning

confidence: 99%

The Sensitized Bioluminescence Mechanism of Bacterial Luciferase

Lee

Müller²,

Visser

2018

Photochem & Photobiology

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After more than one‐half century of investigations, the mechanism of bioluminescence from the FMNH2 assisted oxygen oxidation of an aliphatic aldehyde on bacterial luciferase continues to resist elucidation. There are many types of luciferase from species of bioluminescent bacteria originating from both marine and terrestrial habitats. The luciferases all have close sequence homology, and in vitro, a highly efficient light generation is obtained from these natural metabolites as substrates. Sufficient exothermicity equivalent to the energy of a blue photon is available in the chemical oxidation of the aldehyde to the corresponding carboxylic acid, and a luciferase‐bound FMNH‐OOH is a key player. A high energy species, the source of the exothermicity, is unknown except that it is not a luciferin cyclic peroxide, a dioxetanone, as identified in the pathway of the firefly and the marine bioluminescence systems. Besides these natural substrates, variable bioluminescence properties are found using other reactants such as flavin analogs or aldehydes, but results also depend on the luciferase type. Some rationalization of the mechanism has resulted from spatial structure determination, NMR of intermediates and dynamic optical spectroscopy. The overall light path appears to fall into the sensitized class of chemiluminescence mechanism, distinct from the dioxetanone types.

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Time‐Resolved Fluorescence Study of the Dissociation of FMN from the Yellow Fluorescence Protein from Vibrio fischeri

Cited by 23 publications

References 19 publications

Structural Dynamics of Green Fluorescent Protein Alone and Fused with a Single Chain Fv Protein

Structural Dynamics of Green Fluorescent Protein Alone and Fused with a Single Chain Fv Protein

Protein-protein complexation in bioluminescence

The Sensitized Bioluminescence Mechanism of Bacterial Luciferase

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