Green‐fluorescent protein mutants with altered fluorescence excitation spectra

Ehrig, Torsten; O’Kane, Dennis J.; Prendergast, Franklyn G.

doi:10.1016/0014-5793(95)00557-p

Cited by 152 publications

(98 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The absence of Glu-222 or its displacement from the position standard in GFPs correlates with the excitation peak at ϳ480 nm, which is presumably due to the deprotonated state of the chromophore (27). Indeed, replacement of Glu-222 with a Gly in WT avGFP shifts the excitation maximum in spectra from 398 to 481 nm, leaving the emission peak at ϳ507 nm (28). On the other hand, the proposed restoration of the conventional position of Glu-222 side chain in aceGFP-G222E-Y220L shifts the excitation peak from 480 to 390 nm, characteristic for WT avGFP (28) (Fig.…”

Section: Discussionmentioning

confidence: 96%

Structural Evidence for a Dehydrated Intermediate in Green Fluorescent Protein Chromophore Biosynthesis

Pletneva

Плетнев

Lukyanov

et al. 2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

The acGFPL is the first-identified member of a novel, colorless and non-fluorescent group of green fluorescent protein (GFP)-like proteins. Its mutant aceGFP, with Gly replacing the invariant catalytic Glu-222, demonstrates a relatively fast maturation rate and bright green fluorescence ( ex ‫؍‬ 480 nm, em ‫؍‬ 505 nm). The reverse G222E single mutation in aceGFP results in the immature, colorless variant aceGFP-G222E, which undergoes irreversible photoconversion to a green fluorescent state under UV light exposure. Here we present a high resolution crystallographic study of aceGFP and aceGFP-G222E in the immature and UV-photoconverted states. A unique and striking feature of the colorless aceGFP-G222E structure is the chromophore in the trapped intermediate state, where cyclization of the protein backbone has occurred, but Tyr-66 still stays in the native, non-oxidized form, with C ␣ and C ␤ atoms in the sp 3 hybridization. This experimentally observed immature aceGFP-G222E structure, characterized by the non-coplanar arrangement of the imidazolone and phenolic rings, has been attributed to one of the intermediate states in the GFP chromophore biosynthesis. The UV irradiation ( ‫؍‬ 250 -300 nm) of aceGFP-G222E drives the chromophore maturation further to a green fluorescent state, characterized by the conventional coplanar bicyclic structure with the oxidized double Tyr-66 C ␣ ‫؍‬C ␤ bond and the conjugated system of -electrons. Structurebased site-directed mutagenesis has revealed a critical role of the proximal Tyr-220 in the observed effects. In particular, an alternative reaction pathway via Tyr-220 rather than conventional wild type Glu-222 has been proposed for aceGFP maturation.Green fluorescent proteins (GFP) 2 and the GFP-like proteins (FPs) have become in recent years very useful tools in many areas of cell biology, biotechnology, and medicine. These proteins exhibit a wide spectral range of fluorescence, from blue to far-red. It became possible to effectively use FPs as single or coupled biomarkers for multicolor labeling of proteins, subcellular compartments, and specific tissue regions. Utilization of FPs enabled monitoring of a variety of characteristics, such as cellular pH and ion concentration, tracking of expression, intracellular localization, and trafficking of proteins of interest in the cell or whole organism and following their interactions with other cellular components (1-5).Chromoproteins are another large group of non-fluorescent counterparts of FPs that share with them the principal fold but not spectral properties (6, 7). However, a number of artificially created, genetically engineered variants of chromoproteins do exhibit fluorescence (6, 8). Members of both fluorescent and non-fluorescent families possess visible coloration corresponding to an absorption range of 450 -610 nm. Extensive diversity of their photophysical characteristics arises mostly from variations in the chemical structure of the internal chromophore group and in the stereochemistry of its adjacent environment.The ...

show abstract

Section: Discussionmentioning

confidence: 96%

Structural Evidence for a Dehydrated Intermediate in Green Fluorescent Protein Chromophore Biosynthesis

Pletneva

Плетнев

Lukyanov

et al. 2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…This result is also at odds with the mechanical compression hypothesis, in which the initial cyclization reaction is thermodynamically favorable. Further, our architecturally driven conjugation-trapping mechanism accounts for the robustness of chromophore maturation despite many mutations at or surrounding the chromophore (4), including R96A and E222G (43), and provides a scaffold for specific functional group chemistry to accelerate chromophore maturation. Toward that end, we suggest that conserved residues R96 and E222 have primarily electrostatic roles in chromophore formation and the T62 carbonyl oxygen is the base that initiates the dehydration reaction (Fig.…”

Section: Discussionmentioning

confidence: 99%

Mechanism and energetics of green fluorescent protein chromophore synthesis revealed by trapped intermediate structures

Barondeau

Putnam

Kassmann

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

200

266

View full text Add to dashboard Cite

Green fluorescent protein has revolutionized cell labeling and molecular tagging, yet the driving force and mechanism for its spontaneous fluorophore synthesis are not established. Here we discover mutations that substantially slow the rate but not the yield of this posttranslational modification, determine structures of the trapped precyclization intermediate and oxidized postcyclization states, and identify unanticipated features critical to chromophore maturation. The protein architecture contains a dramatic Ϸ80°bend in the central helix, which focuses distortions at G67 to promote ring formation from amino acids S65, Y66, and G67. Significantly, these distortions eliminate potential helical hydrogen bonds that would otherwise have to be broken at an energetic cost during peptide cyclization and force the G67 nitrogen and S65 carbonyl oxygen atoms within van der Waals contact in preparation for covalent bond formation. Further, we determine that under aerobic, but not anaerobic, conditions the Gly-Gly-Gly chromophore sequence cyclizes and incorporates an oxygen atom. These results lead directly to a conjugation-trapping mechanism, in which a thermodynamically unfavorable cyclization reaction is coupled to an electronic conjugation trapping step, to drive chromophore maturation. Moreover, we propose primarily electrostatic roles for the R96 and E222 side chains in chromophore formation and suggest that the T62 carbonyl oxygen is the base that initiates the dehydration reaction. Our molecular mechanism provides the basis for understanding and eventually controlling chromophore creation.T he Aequorea victoria green fluorescent protein (GFP) undergoes a remarkable posttranslational modification to create a chromophore out of its amino acids (S65, Y66, and G67) (1-3). GFP is small (238 aa), tolerates both N-and C-terminal fusions, and can be targeted to specific cellular locations (4). Synthesis of the GFP fluorophore occurs spontaneously after protein folding without cofactors or accessory proteins (5), making GFP-protein fusions tractable in a variety of organisms. GFP mutants and homologs exhibit fluorescent emission maxima ranging from blue to red (3, 6-8), 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 (9), pH (10, 11), metal ion concentrations (12, 13), and halide levels (14, 15). 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 (4, 16). The crystallographic structure of GFP reveals that the overall fold is an 11-stranded antiparallel ␤-barrel protein with the chromophore located near the geometric center of the barrel on a distorted ␣-helix (1, 17). Few molecular details are known about chromophore maturation.The proposed fluorophore formation mechanism entails three steps: ...

show abstract

“…Excitation at either of the two wavelengths results in emission of green light at 508 nm (for photophysical and photochemical details see review [2]). These fluorescence properties have been changed by genetic engineering leading to several mutants [8][9][10][11][12]. Two of these mutants are of special interest for cell biologists.…”

Section: Properties Of Wildtype and Mutant Gfpsmentioning

confidence: 99%

Green fluorescent protein: applications in cell biology

1996

View full text Add to dashboard Cite

The green fluorescent protein (GFP) of Aequorea victoria is a unique in vivo reporter for monitoring dynamic processes in cells or organisms. As a fusion tag GFP can be used to localize proteins, to follow their movement or to study the dynamics of the subcellular compartments to which these proteins are targeted. Recent studies where GFP technology has revealed new insights regarding physiological activities of living cells are discussed.

show abstract

Green‐fluorescent protein mutants with altered fluorescence excitation spectra

Cited by 152 publications

References 18 publications

Structural Evidence for a Dehydrated Intermediate in Green Fluorescent Protein Chromophore Biosynthesis

Structural Evidence for a Dehydrated Intermediate in Green Fluorescent Protein Chromophore Biosynthesis

Mechanism and energetics of green fluorescent protein chromophore synthesis revealed by trapped intermediate structures

Green fluorescent protein: applications in cell biology

Contact Info

Product

Resources

About