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Chromogenic cross-linkformation in green fluorescent protein.

Despite its dimeric structure, TurboGFP performs well in some fusions. However, for protein labeling applications we recommend using specially optimized monomeric .

GFP numbering, and indicate a XYG tripeptide,where X is variable, Y is an aromatic amino acid (tyrosine in naturallyoccurring proteins) and G illustrates the amino acid glycine, which is strictlyconserved, meaning it crucial for achieving fluorescence (6).

EGFPs are GFPproteins that have been altered to produce different properties.

Synthesis of the chromophore of the ..

Through the discussion of GFP's structure and function in terms of theY66L substitution,

The proposed fluorophore formation mechanism entails three steps: peptide cyclization initiated by nucleophilic attack of the G67 amide nitrogen atom on the S65 carbonyl carbon to create a five-membered imidazolone ring, dehydration of the S65 carbonyl oxygen, and rate-limiting oxidation of the Y66 Cα—Cβ bond to conjugate the ring systems (, , ). The enzyme histidine ammonia lyase (HAL) undergoes a related posttranslational modification to generate an electrophile from the tripeptide loop sequence Ala-Ser-Gly (). There are currently two proposals for the driving force of peptide cyclization in each system: the mechanical compression hypothesis, which suggests that cyclization relaxes an energetically unfavorable precyclized state (, ), and the alternative model where either tyrosine oxidation (GFP) () or serine dehydration (HAL) () precedes cyclization. In GFP, conserved residues R96 and E222 have been proposed to have key roles in chromophore synthesis but have not been experimentally evaluated. This lack of experimental data is likely caused by the difficulties of investigating posttranslational modification reactions that occur during or rapidly after protein folding.

The Aequorea victoria green fluorescent protein (GFP) undergoes a remarkable posttranslational modification to create a chromophore out of its amino acids (S65, Y66, and G67) (–). GFP is small (238 aa), tolerates both N- and C-terminal fusions, and can be targeted to specific cellular locations (). Synthesis of the GFP fluorophore occurs spontaneously after protein folding without cofactors or accessory proteins (), making GFP-protein fusions tractable in a variety of organisms. GFP mutants and homologs exhibit fluorescent emission maxima ranging from blue to red (, –), which allow concurrent surveillance of multiple targets. Together, these properties have fundamentally altered in vivo molecular tagging and cell labeling. In addition, GFP-based indicators monitor cellular redox potential (), pH (, ), metal ion concentrations (, ), and halide levels (, ). Because of these applications and the novel fluorophore, there have been extensive structural, spectroscopic, and biochemical characterizations of the protein and its mutants, all in the mature chromophore state (, ). The crystallographic structure of GFP reveals that the overall fold is an 11-stranded antiparallel β-barrel protein with the chromophore located near the geometric center of the barrel on a distorted α-helix (, ). Few molecular details are known about chromophore maturation.

Variations on the GFP Chromophore - Journal of …

Here we report mutations that substantially slow chromophore formation, determine GFP structures in trapped pre- and oxidized postcyclization states, identify unanticipated features critical for this posttranslational modification, and propose additional functional roles for residues R96 and E222 and the carbonyl oxygen of T62. These discoveries lead directly to a conjugation-trapping mechanism for GFP fluorophore synthesis.

Green fluorescent proteins havecaused expansions in all sorts of fields.

The name "greenfluorescent protein" was given to the second protein that is responsible forthe absorption of the blue light and its conversion into a lower-energy greenlight.

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Gfp chromophore synthesis essay - Marc Randolph


In green fluorescent protein (GFP), ..

Cell-free synthesis, a method for the rapid expression of proteins, is increasingly used to study interactions of complex biological systems. GFP and its variants have become indispensable for fluorescence studies in live cells and are equally attractive as reporters for cell-free systems. This work investigates the use of fluorescence fluctuation spectroscopy (FFS) as a tool for quantitative analysis of protein interactions in cell-free expression systems. We also explore chromophore maturation of fluorescent proteins, which is of crucial importance for fluorescence studies. A droplet sample protocol was developed that ensured sufficient oxygenation for chromophore maturation and ease of manipulation for titration studies. The kinetics of chromophore maturation of EGFP, EYFP, and mCherry were analyzed as a function of temperature. A strong increase in the rate from room temperature to 37 °C was observed. We further demonstrate that all EGFP proteins fully mature in the cell-free solution and that brightness is a robust parameter specifying stoichiometry. Finally, FFS is applied to study the stoichiometry of the nuclear transport factor 2 in a cell-free system over a broad concentration range. We conclude that combining cell-free expression and FFS provides a powerful technique for quick, quantitative study of chromophore maturation and protein-protein interaction.

Syntheses of highly fluorescent GFP-chromophore …

TurboGFP is an improved variant of the green fluorescent protein cloned from copepod (Arthropoda; Crustacea; Maxillopoda; Copepoda) [Shagin , 2004]. It possesses bright green fluorescence (excitation/ emission max = 482/ 502 nm) that is visible earlier than fluorescence of other green fluorescent proteins.

Результаты поиска по "Green Fluorescent Protein"

N2 - Cell-free synthesis, a method for the rapid expression of proteins, is increasingly used to study interactions of complex biological systems. GFP and its variants have become indispensable for fluorescence studies in live cells and are equally attractive as reporters for cell-free systems. This work investigates the use of fluorescence fluctuation spectroscopy (FFS) as a tool for quantitative analysis of protein interactions in cell-free expression systems. We also explore chromophore maturation of fluorescent proteins, which is of crucial importance for fluorescence studies. A droplet sample protocol was developed that ensured sufficient oxygenation for chromophore maturation and ease of manipulation for titration studies. The kinetics of chromophore maturation of EGFP, EYFP, and mCherry were analyzed as a function of temperature. A strong increase in the rate from room temperature to 37 °C was observed. We further demonstrate that all EGFP proteins fully mature in the cell-free solution and that brightness is a robust parameter specifying stoichiometry. Finally, FFS is applied to study the stoichiometry of the nuclear transport factor 2 in a cell-free system over a broad concentration range. We conclude that combining cell-free expression and FFS provides a powerful technique for quick, quantitative study of chromophore maturation and protein-protein interaction.

Green fluorescent protein research papers

AB - Cell-free synthesis, a method for the rapid expression of proteins, is increasingly used to study interactions of complex biological systems. GFP and its variants have become indispensable for fluorescence studies in live cells and are equally attractive as reporters for cell-free systems. This work investigates the use of fluorescence fluctuation spectroscopy (FFS) as a tool for quantitative analysis of protein interactions in cell-free expression systems. We also explore chromophore maturation of fluorescent proteins, which is of crucial importance for fluorescence studies. A droplet sample protocol was developed that ensured sufficient oxygenation for chromophore maturation and ease of manipulation for titration studies. The kinetics of chromophore maturation of EGFP, EYFP, and mCherry were analyzed as a function of temperature. A strong increase in the rate from room temperature to 37 °C was observed. We further demonstrate that all EGFP proteins fully mature in the cell-free solution and that brightness is a robust parameter specifying stoichiometry. Finally, FFS is applied to study the stoichiometry of the nuclear transport factor 2 in a cell-free system over a broad concentration range. We conclude that combining cell-free expression and FFS provides a powerful technique for quick, quantitative study of chromophore maturation and protein-protein interaction.

The Journal of Organic Chemistry (ACS Publications)

Once amino acid residues correctly fold, threestages for GFP chromophore synthesis are required: cyclization of thetripeptide, oxidation to cyclic imine, and dehydration of the Cα- Cβbond of Tyr66 which results in the mature chromophore that has the ability toemit green light (5).

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