Max-Planck-Institut für biophysikalische Chemie Karl-Friedrich-Bonhoeffer-Institut
MPIbpc11. Jahrgang Göttingen AusgabeNews Nr. 12 Dezember 2005
Berichte aus den Abteilungen Insight into the structure and mechanism of the reversible photoswitch of a fl uorescent protein A multi-departmental research approach
Martin Andresen1, Markus C. Wahl2, André C. Stiel1, Frauke Gräter3, Lars V. Schäfer3, Simon Trowitzsch2, Gert Weber2, Christian Eggeling1, Helmut Grubmüller3, Stefan W. Hell1 and Stefan Jakobs1 1 Dept. of NanoBiophotonics 2 Dept. of Cellular Biochemistry/Macromolecular X-Ray Crystallography 3 Dept. of Theoretical and Computational Biophysics
Structure and mechanism of a molecular protein. However, whereas the ‘classical’ light switch fl uorescent proteins are practically always Fluorescent proteins, like the well in their on-state, until they are irreversibly known green fl uorescent protein (GFP) are bleached, the switchability of asFP595 and Zusammenfassung widely used to generate fusion tags that act Dronpa may provide the tools to get new Schaltbare Proteine, die sich durch Be- as fl uorescent reporters for studying protein insights into the diffusive or directed move- strahlung mit sichtbarem Licht reversibel localizations and dynamics in live cells. ment of the host proteins. The intriguing zwischen einem fl uoreszierenden ‚Ein’- Recently, with asFP595 isolated from the features of the RSFPs make many other und einem nicht-fl uoreszierenden ‚Aus’- sea anemone Anemonia sulcata (Fig. 1) and applications imaginable. For example, the Zustand hin und herschalten lassen, sind Dronpa cloned from the coral Pectiniidae, reversible photoswitching of fl uorescent erst seit wenigen Jahren bekannt. Derzeit the fi rst two members of the new class of markers may provide nanoscale resolution Fortsetzung auf Seite 3 reversibly switchable fl uorescent proteins in fl uorescence microscopy using lenses (RSFP) have been described. RSFPs can and regular illumination. be transferred by light irradiation from a The RSFP asFP595, used in our studies, non-fl uorescent ‘off’ to a fl uorescent ‘on’ has some favorable switching properties: state and back again. (i) Switching from a non-fl uorescent off- Impressum Somewhat related fl uorescent proteins, state to a fl uorescent on-state is induced namely photoactivatable proteins like PA- by irradiation with green light. (ii) Red Herausgeber: GFP or PS-CFP, that can be irreversibly fl uorescence emission of the on-state is Presse- und Öffentlichkeitsarbeit switched by light from one state into a dif- elicited by the same green light. And (iii)
found that asFP595-A143S protein crys- tals can be reversibly transferred by light between the on- and the off-state. Non-ir- radiated asFP595-A143S crystals are barely fl uorescent, whereas after irradiation with green light, they display bright red fl uo- rescence that can be switched off with blue light (Fig. 3). Again, this switching is reversible numerous times. The switching kinetics of the protein in solution is very similar to the kinetics of the protein within the crystal lattice. The crystallized proteins thus behave as in solution. Therefore crys- tals were irradiated for 0, 1, or 5 minutes with green light and subsequently cap- tured by fl ash freezing in liquid nitrogen to obtain structures of the protein in its off- and on-states. We found that the on- and the off-state from the Snakelocks anemone, is far from The on- and the off-state chromophores adopt different conforma- being an optimal tag. Therefore optimi- In order to understand the structural tions: The chromophores of the irradiated zation of this tag is needed. To this end, differences between the on- and the off- proteins (on-state) were in a cis-confor- researchers from three departments of this state, we used the protein variant asFP595- mation, whereas the those of the non- institute set out in a collaborative effort to A143S, which exhibits slower switching irradiated proteins (off-state) adopted a understand the structure and the switching kinetics than the wild type protein. We trans-conformation (Fig. 4). As expected, mechanism of asFP595.
Structure of asFP595 First, we determined the atomic off-state structure of asFP595. Large amounts of the protein were produced in bacteria, purifi ed and crystallized. After solving the x-ray structure, we (and independently two other groups) discovered that its structure is very similar to that of GFP. Like many other fl uorescent proteins, asFP595 forms tetram- ers. It is a globular protein, with a very rigid structure, so that the chromophoric region is shielded from the environment. The chromophore resides in a helical seg- ment that is enclosed by a β-barrel (Fig. 2). The chromophore is formed by three amino acids (M63-Y64-G65) that rearrange to a conjugated system with a p-hydroxyphenyl and an imidazolinone ring that are con- nected by a methine bridge. Remarkably, in asFP595, the protein backbone is broken at the chromophore. This backbone break is unique for a fl uorescent protein and it Fig. 2. Overall structure of asFP595. A schematic ribbon representation of the quaternary tetrameric appears that this break provides the pro- structure of asFP595 showing the four molecules (A-D) in different colors and the chromophores tein with the intrinsic fl exibility to allow highlighted in yellow. switching of the chromophore with low Abb. 2: Gesamtstruktur von asFP595. In der schematischen Darstellung des tetrameren Proteins sind light energies. die vier Moleküle (A-D) in verschieden Farben dargestellt. Die Chromophore sind gelb eingefärbt.
Seite 2 Fortsetzung ... sind mit den Proteinen asFP595 und Dron- pa erst zwei Vertreter solcher reversibel schaltbaren fl uoreszierenden Proteine (RSFPs) beschrieben worden. Nichts- destotrotz verspricht diese neue Protein- klasse aufgrund ihrer einzigartigen Eigen- schaften bereits jetzt eine Vielzahl interes- santer Anwendungsmöglichkeiten. Das Protein asFP595, dessen Schalt- mechanismus wir in dieser Studie unter- sucht haben, kommt normalerweise in den Tentakelspitzen der Wachsrose Anemonia sulcata vor, einer Seeanemone, die in den lichtdurchfl uteten Flachwasser- bereichen des Mittelmeers und des Nord- atlantiks lebt. asFP595 wird durch Be- leuchtung mit grünem Licht von einem nicht-fl uoreszierenden ‚Aus’-Zustand in Fig. 3. (a) Reversible photoswitching of an asFP595-A143S protein crystal under a fl uorescence microscope. Irradiation with green light (g= 550 ± 20 nm) yields brightly fl uorescent crystals. The einen fl uoreszierenden ‚Ein’-Zustand ver- fl uorescence is quenched by gentle irradiation with blue light (b= 450 ± 20 nm). (b) Brightfi eld setzt. Von diesem ‚Ein’-Zustand fällt es image of the crystal. Scale bar: 20µm. spontan in den ‚Aus’-Zustand zurück, Abb. 3: (a) Reversibles Photoschalten von asFP595-A143S Proteinkristallen mit Hilfe eines kann aber auch durch Beleuchtung mit Fluoreszenzmikroskops. Bestrahlung mit grünem Licht (g= 550 ± 20 nm) ergibt stark fl uoreszierende blauem Licht zurückgeschaltet werden. Kristalle. Die Fluoreszenz kann durch schwache Bestrahlung mit blauem Licht (b= 450 ± 20 nm) Dieses lichtgetriebene Schalten ist rever- gelöscht werden. (b) Durchlichtbild des Proteinkristalls. Maßstab: 20µm. sibel und viele Male wiederholbar. In einer gemeinsamen Studie dreier Abteilungen in the briefl y irradiated crystals, we identi- visible in the crystal structure, but it is haben wir die molekulare Struktur von fi ed a mixture of chromophores of both likely a consequence of the changes in asFP595 bestimmt und wesentliche Teile conformations. the immediate protein surrounding after des Schaltmechanismus aufgeklärt. Diese Spectroscopic data further indicates that a trans-cis-isomerization. Untersuchungen haben gezeigt, dass nach in the cis-conformation, the chromophoric Hence, from this crystallographic der Absorption eines grünen Photons das p-hydroxyphenyl group is in equilibrium analysis, we knew the structural differ- asFP595-Chromophor von einer Trans- in between a (non-fl uorescent) protonated ence between the protein in the on- and eine Cis-Position isomerisiert. Molekular- and a (fl uorescent) non-protonated form. the off-state. As a next step, we set out dynamik-Rechnungen deuten an, dass das Such a proton transfer would not have been to investigate the molecular movements Chromophor bei dieser lichtinduzierten Reaktion eine ‚Hula-Twist’-Bewegung macht. Bei dieser Bewegung dreht sich in erster Linie die Methin-Brücke, wel- che die beiden aromatischen Ringe des Chromophors verknüpft. Tatsächlich ändert das Chromophor während des Hula-Twists seine Position lediglich um 3 x 10-10 Meter. Diese winzige Änderung reicht aus, um aus dem nicht-fl uoreszierenden ein fl uo- reszierendes Protein zu machen. Diese neuen Erkenntnisse über die Struktur und den Schaltmechanismus des Prote- ins sollten es zukünftig ermöglichen, mit Hilfe von gerichteten molekularbiologi- Fig. 4. Trans- and cis-conformation of the MYG chromophore in the ‘off’- and the ‘on’-state, respec- schen Ansätzen das Protein für weitere tively. Atoms are color-coded by atom type (carbon – salmon, oxygen – red, nitrogen – blue). Final Anwendungen zu optimieren. Denkbare 2Fo-Fc electron densities around the chromophores are contoured at the 1σ level. For asFP595- Einsatzbereiche eines solchen modifi zier- A143S irradiated for one minute, both isomerization states are depicted because both structures were ten asFP595 reichen von der höchstauf- observed in a single crystal with equal proportions. lösenden Fluoreszenzmikroskopie bis hin Abb. 4: Trans- und Cis-Konformation des MYG Chromophors im ‘Aus’- und im ‘An’-Zustand. Die zum Einsatz als Datenspeicher. Atome sind farbkodiert dargestellt (Kohlenstoff – lachsfarben, Sauerstoff – rot, Stickstoff – blau). Die 2Fo-Fc Elektronendichten um die Chromophoren sind auf dem 1σ-Niveau abgebildet. Im Falles des für eine Minute belichteten asFP595-A143S Kristalls sind beide Konformationen dargestellt, da beide Strukturen zu gleichen Anteilen im Kristall bestimmt wurden.
Seite 3 the difference between a fl uorescent and References a non-fl uorescent asFP595. Andresen, M., M.C. Wahl, A.C. Stiel, F. Gräter, L.V. Schäfer, S. Trowitzsch, G. Weber, C. Conclusion and Outlook Eggeling, H. Grubmüller, S.W. Hell, and S. Jakobs. 2005. Structure and mechanism of the reversible With this study we have elucidated the photoswitch of a fl uorescent protein. Proc. Natl. overall molecular light induced switching Acad. Sci. USA. 102:13070-13074. mechanism of the remarkable protein M. Hofmann, C. Eggeling, S. Jakobs, S.W. asFP595. It exhibits a complex photo- Hell. Breaking the diffraction barrier in fl uo- Fig. 5. Hula twist mechanism. Upon irradiation physical behavior, so it may very well be rescence microscopy at low light intensities with green light the chromophore changes its anticipated that a more detailed under- by using reversibly photoswitchable pro- conformation from the non-fl uorescent trans-con- teins. Proc. Natl. Acad. Sci. USA. In press. standing of its mechanism will bear even formation (blue) into the fl uorescent cis-confor- Lukyanov, K.A., A.F. Fradkov, N.G. Gurskaya, mation (red) with a so-called hula twist. In a hula more surprises. Nonetheless, the obtained M.V. Matz, Y.A. Labas, A.P. Savitsky, M.L. twist mechanism, the two rings of the chromo- insight provides a solid framework of this Markelov, A.G. Zaraisky, X.N. Zhao, Y. Fang, W.Y. phore almost stay in place and only the methine molecular light switch for making it pos- Tan, and S.A. Lukyanov. 2000. Natural animal bridge that connects both rings moves. sible to lay out strategies for semi-rational coloration can be determined by a nonfl uores- Abb. 5: „Hula Twist“-Mechanismus. Bei der licht- approaches to engineer asFP595 variants cent green fl uorescent protein homolog. J. Biol. Chem. 275:25879-25882. induzierten Konformationsänderung des Chromo- with improved properties. Mutants of phors aus der nicht-fl uoreszierenden Trans-Form (blau) in die fl uoreszierende Cis-Form (rot) dreht asFP595 with better switching properties sich in erster Linie die Methin-Brücke, welche and higher quantum effi ciency may be Stefan Jakobs die beiden aromatischen Ringe des Chromophors used for applications ranging from high Studied biology in Kaisers- verknüpft. resolution optical microscopy to data lautern and Manchester. storage. After fi nishing his PhD at of the chromophore that take place upon the MPI for Plant Breeding irradiation to induce a trans to cis isomeri- Research in Cologne and zation. work at the John Innes Centre in Norwich, he joined in 1999 the Computer simulations of the switching High Resolution Microscopy Group, which mechanism The team: From left to right. became the Dept. of NanoBiophotonics in To this end we performed molecular Top row: Stefan Jakobs, André C. Stiel, Lars V. 2002. Since June 2005 he is heading the dynamics (MD) and free dynamics simu- Schäfer, Frauke Gräter, Helmut Grubmüller. research group ‘Mitochondrial Structure lations. The basis for these calculations Bottom row: Gert Weber, Simon Trowitzsch, and Dynamics’. were the crystal structures of the protein Martin Andresen, Christian Eggeling, Markus C. in the on- and the off-state which represent Wahl, Stefan W. Hell. the start and the fi nal position of the light induced chromophore movement. By MD we simulated the two conceivable trans- cis photoisomerization pathways and also considered intramolecular properties of the chromophore adopting high-level quantum mechanical computations. As an outcome, these computer simulations showed that both the intrinsic chromophore proper- ties, as well as the overall protein con- formation, point to a so-called bottom hula twist mechanism as the predominant isomerization mechanisms in asFP595. In a hula twist mechanism, the two rings of the chromophore almost stay in place and only the methine bridge that connects both rings moves (Fig. 5). Such a hula twist is the most space effi cient way to transform the trans- into a cis-conformation. In fact, the largest movement occurs at the phenolic oxygen of the chromophore, which trans- -10 lates by only 3•10 meter. This infi nitesi- mal movement, which also likely induces the already mentioned protonation event at the phenolic oxygen, seems to make all
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