Structural basis of spectral shifts in the yellow-emission variants of green fluorescent protein

Wachter, Rebekka M.; Elsliger, Marc‐André; Kallio, Karen; Hanson, George T.; Remington, S.J.

doi:10.1016/s0969-2126(98)00127-0

Cited by 409 publications

(546 citation statements)

References 45 publications

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“…These data demonstrate that the YFP molecule, which has two additional cysteines, when fused to the COOH terminus of C5aR does not influence the crosslinking reaction, as evidenced by the nearly binomial distribution of the three C5aR dimer species. It is not surprising that the cysteines in YFP do not participate in cross-linking, since in the crystal structure they are not surface-accessible (40). However, it was important to dismiss YFP as a mediator of C5aR interaction, since it has been shown that GFP molecules can dimerize at high concentrations (41).…”

Section: Resultsmentioning

confidence: 99%

C5a Receptor Oligomerization

Klco

Lassere

Baranski

2003

Journal of Biological Chemistry

108

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G protein-coupled receptors (GPCRs), stimulated by hormones and sensory stimuli, act as molecular switches to relay activation to heterotrimeric G proteins. Recent studies suggest that GPCRs form dimeric or oligomeric structures, a phenomenon that has long been established for growth factor receptors. The elucidation of the domains of GPCRs that mediate receptor association is of critical importance for understanding the function of GPCR oligomers. Using a disulfide-trapping strategy to probe the intermolecular contact surfaces, we demonstrate cross-linking of C5a receptors in membranes prepared from both human neutrophils and stably transfected mammalian cells that is mediated by a cysteine in the second intracellular loop. To explore other surfaces that might be involved in the oligomerization of C5a receptors, we constructed receptors with individual cysteines in other intracellular regions. C5a receptors with a cysteine in the first intracellular loop or the carboxyl terminus displayed the fastest kinetics of dimer formation, whereas an intracellular loop 3 cysteine displayed minimal cross-linking. Since the rate of disulfide trapping reflects the proximity of sulfhydryl groups, assuming similar accessibility and flexibility, these results imply a symmetric dimer interface that may involve either transmembrane helices 1 and 2 or helix 4. However, neither model can account for the ability of the native cysteine in the second intracellular loop to mediate efficient crosslinking. Based on these observations, we propose that C5a receptors form higher order oligomers (i.e. tetramers) or clusters in the membrane.G protein-coupled receptors (GPCRs) 1 are an evolutionarily conserved family of transmembrane receptors that are characterized by seven-transmembrane helixes connected by intracellular loops (ICs) and extracellular loops (1, 2). GPCRs mediate a multitude of physiologic responses and serve as common pharmacological targets; more than half of all pharmacological agents that are currently prescribed target GPCRs (3). Receptor activation is accomplished by a remarkably diverse group of ligands to catalyze the exchange of GTP for GDP on the ␣ subunit of heterotrimeric G proteins. This exchange results in the dissociation of the ␣-GTP subunit from the ␤␥ dimer. Both complexes can then activate downstream targets, such as effector enzymes and ion channels (4, 5).Despite numerous structure-function studies of many different GPCRs, a fundamental question remains: Do GPCRs function as monomers or as oligomers? For the ϳ25 members of class C receptors, the answer is clear. These receptors form dimers through specialized structures, such as disulfidebonded extracellular domains (metabotropic glutamate receptors) (6, 7) or coiled-coil interactions of the carboxyl-terminal tails (GABA B receptors) (8). For the more than 500 members of the rhodopsin family of GPCRs and the 60 members of the secretin family of GPCRs, studies now demonstrate that a rapidly increasing number of these GPCRs form oligomers (9, 10). Early work...

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Section: Resultsmentioning

confidence: 99%

C5a Receptor Oligomerization

Klco

Lassere

Baranski

2003

Journal of Biological Chemistry

108

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“…The chromophore in the YFP-H148G has an exposed phenolic oxygen (22) and would seem available for rapid protonation. The overall pK of YFP-H148G differs only by 1 compared with the 10C variant studied here, where H148 is hydrogen-bonded to the phenolate ion.…”

Section: Discussionmentioning

confidence: 99%

Protonation, Photobleaching, and Photoactivation of Yellow Fluorescent Protein (YFP 10C): A Unifying Mechanism

et al. 2005

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Yellow fluorescent protein (YFP 10C) is widely used as a probe in biology, but its complex photochemistry gives rise to unusual behavior that requires fuller definition. Here we characterize the kinetics of protonation and reversible bleaching over time scales of picoseconds to hours. Stopped-flow and pressure-jump techniques showed that protonation of the fluorescent YFP -anion state is two-step with a slow transition that accounts for blinking of 527 nm emission at the single molecule level on the seconds time scale. Femtosecond spectroscopy revealed that the protonated excited-state (YFPH*) decayed predominantly by a radiationless mechanism, but emission at 460 nm was detected within the first picosecond. Limited excited-state proton transfer leads to 527 nm emission characteristic of the YFP -* anion. Prolonged continuous wave illumination at the peak of YFP -absorbance (514 nm) yields, irreversibly, a weakly fluorescent product that absorbs at 390 nm. This "photobleaching" process also gives a different species (YFPHrb) that absorbs at 350/430 nm and spontaneously regenerates YFP -in the dark on the time scale of hours but can be photoactivated by UV light to regenerate YFP -within seconds, via a ground-state protonated intermediate. Using a pulsed laser for photobleaching resulted in decarboxylation of YFP as indicated by the mass spectrum. These observations are accounted for in a unifying kinetic scheme.Green fluorescent protein (GFP) 1 and its color variants have found wide use in biology to probe protein dynamics in vitro and within cells (1). They have also attracted the attention of spectroscopists owing to their complex photochemistry (2-5). In particular, those in the YFP class (i.e. containing a T203Y or T203F mutation to shift the emission to 527 nm) have been shown to undergo fluctuations of emission intensity over a wide range of times scales and reversible photobleaching (6-8). Fluctuations in emission were observed by wide field microscopy at the single molecule level by immobilizing individual YFP molecules in acrylamide or agarose gels and using TIRF excitation (6, 9). Molecules were found to blink on the seconds time scale at low laser powers (<500 W cm -2 ). Increasing laser power reduced the lifetime of the emitting state, but not in direct proportion to the laser power. Initial studies discussed the possibility that the fluctuations were related to the protonation of the phenolate moiety of the fluorophore derived from Y66 (6), but later studies found little pH dependence of the onand off-times when monitored at a laser power of 5000 W cm -2 (9).Members of the YFP class also showed a photochromic switching behavior. Molecules that were bleached by 488 nm irradiation, could be induced to fluoresce by illumination with near UV or blue light (6-8). This reversible photobleaching reaction of YFP was observed in FRET measurements (10). Illumination of YFP at 514 nm resulted in bleaching of the YFP acceptor and dequenching of a donor CFP molecule. Once the YFP was bleached, illumina...

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“…The aromatic ring of the His 197 is highly polarizable and would help stabilize the excited state of the chromophore. It has been proposed that -stacking interactions and increased polarizability of the chromophore are responsible for the significant red-shifted spectra observed for yellow fluorescent variants of GFP from A. victoria, containing aromatic substitutions at Thr 203 (23). Replacement of Thr 203 with histidine, tyrosine, or phenylalanine results in a shift of the maximum of emission of 13-17 nm relative to the GFP variant S65T (24,25).…”

Section: Discussionmentioning

confidence: 99%

The 2.0-Å Crystal Structure of eqFP611, a Far Red Fluorescent Protein from the Sea Anemone Entacmaea quadricolor

Petersen

Wilmann

Beddoe

et al. 2003

Journal of Biological Chemistry

169

189

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We have crystallized and subsequently determined to 2.0-Å resolution the crystal structure of eqFP611, a far red fluorescent protein from the sea anemone Entacmaea quadricolor. The structure of the protomer, which adopts a ␤-can topology, is similar to that of the related monomeric green fluorescent protein (GFP). The quaternary structure of eqFP611, a tetramer exhibiting 222 symmetry, is similar to that observed for the more closely related red fluorescent protein DsRed and the chromoprotein Rtms5. The unique chromophore sequence (Met 63 -Tyr 64 -Gly 65 ) of eqFP611, adopts a coplanar and trans conformation within the interior of the ␤-can fold. Accordingly, the eqFP611 chromophore adopts a significantly different conformation in comparison to the chromophore conformation observed in GFP, DsRed, and Rtms5. The coplanar chromophore conformation and its immediate environment provide a structural basis for the far red, highly fluorescent nature of eqFP611. The eqFP611 structure extends our knowledge on the range of conformations a chromophore can adopt within closely related members of the green fluorescent protein family.

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Structural basis of spectral shifts in the yellow-emission variants of green fluorescent protein

Cited by 409 publications

References 45 publications

C5a Receptor Oligomerization

C5a Receptor Oligomerization

Protonation, Photobleaching, and Photoactivation of Yellow Fluorescent Protein (YFP 10C): A Unifying Mechanism

The 2.0-Å Crystal Structure of eqFP611, a Far Red Fluorescent Protein from the Sea Anemone Entacmaea quadricolor

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