The 1.7 Å Crystal Structure of Dronpa: A Photoswitchable Green Fluorescent Protein

Wilmann, P.G.; Turcic, Kristina; Battad, Jion; Wilce, Matthew Charles James; Devenish, Rodney J.; Prescott, Mark; Rossjohn, Jamie

doi:10.1016/j.jmb.2006.08.089

Cited by 77 publications

(93 citation statements)

References 45 publications

(53 reference statements)

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“…More recently, crystal structures have been reported for Dronpa in the fluorescent state (30,32). In each case, a slightly noncoplanar cis chromophore was observed, with conserved side chains forming an environment that was virtually superimposable upon that of the mTFP0.7 fluorescent state.…”

Section: Discussionmentioning

confidence: 99%

“…In each case, a slightly noncoplanar cis chromophore was observed, with conserved side chains forming an environment that was virtually superimposable upon that of the mTFP0.7 fluorescent state. Wilmann et al (32) proposed a model for photoswitching of Dronpa that does not involve isomerization, but rather hinges on light-induced protonation of the cis isomer, which seems unlikely. On the other hand, Stiel et al (30) proposed that photoswitching in Dronpa is caused by cis to trans isomerization, accompanied by conformational changes in the side chain of Arg-66 and possibly other residues.…”

Section: Discussionmentioning

confidence: 99%

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Structural basis for reversible photobleaching of a green fluorescent protein homologue

Henderson

Campbell

et al. 2007

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

219

208

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Fluorescent protein (FP) variants that can be reversibly converted between fluorescent and nonfluorescent states have proven to be a catalyst for innovation in the field of fluorescence microscopy. However, the structural basis of the process remains poorly understood. High-resolution structures of a FP derived from Clavularia in both the fluorescent and the light-induced nonfluorescent states reveal that the rapid and complete loss of fluorescence observed upon illumination with 450-nm light results from cistrans isomerization of the chromophore. The photoinduced change in configuration from the well ordered cis isomer to the highly nonplanar and disordered trans isomer is accompanied by a dramatic rearrangement of internal side chains. Taken together, the structures provide an explanation for the loss of fluorescence upon illumination, the slow light-independent recovery, and the rapid light-induced recovery of fluorescence. The fundamental mechanism appears to be common to all of the photoactivatable and reversibly photoswitchable FPs reported to date.crystallography ͉ fluorescence ͉ photoswitching ͉ protein structure G reen fluorescent protein (GFP) from the jellyfish Aequorea victoria (1) and more recently, cyan, yellow, and red fluorescent proteins (FPs), isolated from coral reef organisms (2), have become standard research tools in cellular biology. Structural studies have revealed a universally conserved fold for FPs: an 11-stranded ␤-barrel with a central and axial ␣-helix (3, 4). A sequence of three amino acid residues on this central ␣-helix (Ser-65-Tyr-66-Gly-67 in Aequorea GFP) undergoes a series of autocatalytic posttranslational modifications to form the chromophore. Despite overall structural similarity across FPs from a variety of species and a variety of hues, members of the GFP family are quite diverse with respect to their photophysical properties. For example, properties such as emission color, extinction coefficient, quantum yield, and fluorescence lifetime can vary dramatically among FP variants. These properties are determined by both the influence of the protein matrix on the chromophore environment and the particular covalent chemical structure of the chromophore (5).Another photophysical property common to all FPs is wavelength-dependent change in their f luorescence emission properties upon illumination. In most cases, these changes appear to result from irreversible covalent modification of the chromophore structure or its local protein environment. Examples of such permanent changes include light-induced transition from a f luorescent to a nonf luorescent state (photobleaching), transition from a nonf luorescent to a f luorescent state (photoactivation), or a change in excitation or emission wavelength maxima (photoconversion). Photobleaching, photoactivation, and photoconversion are well documented and can be advantageous for f luorescence imaging applications such as in vivo tracking of fusion protein movements (6 -9). A less well documented process, occasionally described as ''re...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Structural basis for reversible photobleaching of a green fluorescent protein homologue

Henderson

Campbell

et al. 2007

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

219

208

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“…Dronpa undergoes photoswitching in the crystalline state, which resulted in the structural determination of the two species. Stiel et al 3 and Wilmann et al 4 determined the X-ray structures of the highly fluorescent on state of Dronpa, whereas Andresen et al 5 determined the off structure. Mizuno et al 6 performed NMR experiments of the photoswitching of Dronpa in solution, establishing photoinduced changes of the b-barrel structure.…”

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confidence: 99%

“…The off-state structure was cryogenically trapped after full conversion of crystals with green illumination 5 . The chromophore of Dronpa is derived from the Cys-Tyr-Gly tripeptide and is present in a cis conformation in the on state 3,4 , as in the A. victoria GFP 7,8 . Substantial structural rearrangements of the protein environment were shown to coincide with the cis-trans photoisomerization of the chromophore.…”

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confidence: 99%

Ground-state proton transfer in the photoswitching reactions of the fluorescent protein Dronpa

et al. 2013

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The reversible photoswitching between the 'on' and 'off' states of the fluorescent protein Dronpa involves photoisomerization as well as protein side-chain rearrangements, but the process of interconversion remains poorly characterized. Here we use time-resolved infrared measurements to monitor the sequence of these structural changes, but also of proton transfer events, which are crucial to the development of spectroscopic contrast. Light-induced deprotonation of the chromophore phenolic oxygen in the off state is a thermal ground-state process, which follows ultrafast (9 ps) trans-cis photoisomerization, and so does not involve excited-state proton transfer. Steady-state infrared difference measurements exclude protonation of the imidazolinone nitrogen in both the on and off states. Pump-probe infrared measurements of the on state reveal a weakening of the hydrogen bonding between Arg66 and the chromophore C ¼ O, which could be central to initiating structural rearrangement of Arg66 and His193 coinciding with the low quantum yield cis-trans photoisomerization

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“…a {(4Z)-2-[1-amino-2-mercaptoethyl]-4-(4-hydroxybenzylidene)-5-oxo-4,5-dihydro-1H-imidazol-1-yl}-acetaldehyde moiety, is formed spontaneously from the tripeptide Cys62-Tyr63-Gly64, referred to as GYC; it is part of an -helix and is positioned in the centre of the -can. It has been demonstrated that cistrans isomerization and protonation and deprotonation of this bicyclic chromophore is a key event in the switching process of Dronpa (Habuchi et al, 2005;Stiel et al, 2007;Wilmann et al, 2006;Andresen et al, 2007). Additionally, certain residues in its vicinity have been shown to have a crucial influence on its photoswitchable properties (Wilmann et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

Structural basis for the influence of a single mutation K145N on the oligomerization and photoswitching rate of Dronpa

Bich

Moeyaert

Hecke

et al. 2012

Acta Crystallogr D Biol Cryst

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Author(s) of this paper may load this reprint on their own web site or institutional repository provided that this cover page is retained. Republication of this article or its storage in electronic databases other than as specified above is not permitted without prior permission in writing from the IUCr.For further information see http://journals.iucr.org/services/authorrights.html Acta Crystallographica Section D: Biological Crystallography welcomes the submission of papers covering any aspect of structural biology, with a particular emphasis on the structures of biological macromolecules and the methods used to determine them. Reports on new protein structures are particularly encouraged, as are structure-function papers that could include crystallographic binding studies, or structural analysis of mutants or other modified forms of a known protein structure. The key criterion is that such papers should present new insights into biology, chemistry or structure. Papers on crystallographic methods should be oriented towards biological crystallography, and may include new approaches to any aspect of structure determination or analysis. Papers on the crystallization of biological molecules will be accepted providing that these focus on new methods or other features that are of general importance or applicability.Crystallography Journals Online is available from journals.iucr.org Acta Cryst. (2012 The crystal structure of the on-state of PDM1-4, a singlemutation variant of the photochromic fluorescent protein Dronpa, is reported at 1.95 Å resolution. PDM1-4 is a Dronpa variant that possesses a slower off-switching rate than Dronpa and thus can effectively increase the image resolution in subdiffraction optical microscopy, although the precise molecular basis for this change has not been elucidated. This work shows that the Lys145Asn mutation in PDM1-4 stabilizes the interface available for dimerization, facilitating oligomerization of the protein. No significant changes were observed in the chromophore environment of PDM1-4 compared with Dronpa, and the ensemble absorption and emission properties of PDM1-4 were highly similar to those of Dronpa. It is proposed that the slower off-switching rate in PDM1-4 is caused by a decrease in the potential flexibility of certain -strands caused by oligomerization along the AC interface.

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The 1.7 Å Crystal Structure of Dronpa: A Photoswitchable Green Fluorescent Protein

Cited by 77 publications

References 45 publications

Structural basis for reversible photobleaching of a green fluorescent protein homologue

Structural basis for reversible photobleaching of a green fluorescent protein homologue

Ground-state proton transfer in the photoswitching reactions of the fluorescent protein Dronpa

Structural basis for the influence of a single mutation K145N on the oligomerization and photoswitching rate of Dronpa

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