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Chromophore protonation state controls photoswitching of the fluoroprotein asFP595.

Schäfer LV, Groenhof G, Boggio-Pasqua M, Robb MA, Grubmüller H - PLoS Comput. Biol. (2008)

Bottom Line: We have identified the tight coupling of trans-cis isomerization and proton transfers in photoswitchable proteins to be essential for their function and propose a detailed underlying mechanism, which provides a comprehensive picture that explains the available experimental data.The structural similarity between asFP595 and other fluoroproteins of interest for imaging suggests that this coupling is a quite general mechanism for photoswitchable proteins.These insights can guide the rational design and optimization of photoswitchable proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Theoretical and Computational Biophysics, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany.

ABSTRACT
Fluorescent proteins have been widely used as genetically encodable fusion tags for biological imaging. Recently, a new class of fluorescent proteins was discovered that can be reversibly light-switched between a fluorescent and a non-fluorescent state. Such proteins can not only provide nanoscale resolution in far-field fluorescence optical microscopy much below the diffraction limit, but also hold promise for other nanotechnological applications, such as optical data storage. To systematically exploit the potential of such photoswitchable proteins and to enable rational improvements to their properties requires a detailed understanding of the molecular switching mechanism, which is currently unknown. Here, we have studied the photoswitching mechanism of the reversibly switchable fluoroprotein asFP595 at the atomic level by multiconfigurational ab initio (CASSCF) calculations and QM/MM excited state molecular dynamics simulations with explicit surface hopping. Our simulations explain measured quantum yields and excited state lifetimes, and also predict the structures of the hitherto unknown intermediates and of the irreversibly fluorescent state. Further, we find that the proton distribution in the active site of the asFP595 controls the photochemical conversion pathways of the chromophore in the protein matrix. Accordingly, changes in the protonation state of the chromophore and some proximal amino acids lead to different photochemical states, which all turn out to be essential for the photoswitching mechanism. These photochemical states are (i) a neutral chromophore, which can trans-cis photoisomerize, (ii) an anionic chromophore, which rapidly undergoes radiationless decay after excitation, and (iii) a putative fluorescent zwitterionic chromophore. The overall stability of the different protonation states is controlled by the isomeric state of the chromophore. We finally propose that radiation-induced decarboxylation of the glutamic acid Glu215 blocks the proton transfer pathways that enable the deactivation of the zwitterionic chromophore and thus leads to irreversible fluorescence. We have identified the tight coupling of trans-cis isomerization and proton transfers in photoswitchable proteins to be essential for their function and propose a detailed underlying mechanism, which provides a comprehensive picture that explains the available experimental data. The structural similarity between asFP595 and other fluoroproteins of interest for imaging suggests that this coupling is a quite general mechanism for photoswitchable proteins. These insights can guide the rational design and optimization of photoswitchable proteins.

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Hydrogen bonding network in the chromophore cavity during force field simulations of zwitterionic chromophores.(A, C) Snapshots from MD simulations of Ztrans and Zcis, respectively. The blue dashed lines indicates the distance between the NH proton of MYG and Glu215, and the red dashed line that between the OH-group of Glu215 and the Nδ atom of His197. (B, D) Time-evolution of the two hydrogen bonds shown in (A) and (C) during representative force field MD simulations.
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pcbi-1000034-g006: Hydrogen bonding network in the chromophore cavity during force field simulations of zwitterionic chromophores.(A, C) Snapshots from MD simulations of Ztrans and Zcis, respectively. The blue dashed lines indicates the distance between the NH proton of MYG and Glu215, and the red dashed line that between the OH-group of Glu215 and the Nδ atom of His197. (B, D) Time-evolution of the two hydrogen bonds shown in (A) and (C) during representative force field MD simulations.

Mentions: To identify possible ESPT pathways, we have carried out extended force field MD simulations of both Ztrans and Zcis and analyzed the relevant hydrogen bonds. Figure 6A shows that, during the simulation of Ztrans, two stable hydrogen bonds were formed between the protonated OH group of Glu215 and His197 as well as between the NH proton of MYG and Glu215. These two hydrogen bonds allow for a proton transfer from Ztrans to the rapidly deactivating Atrans. The OH proton of Glu215 could transfer to the Nδ atom of His197, with a simultaneous or subsequent transfer of the NH proton of the imidazolinone moiety to Glu215. In contrast, during the force field simulation of Zcis, the MYG-Glu215 hydrogen bond remained intact, whereas the Glu215-His197 hydrogen bond broke after about 1 ns (Figure 6B). This differential behavior of Ztrans and Zcis was confirmed by two additional independent MD simulations (data not shown). Based on these results, we assume that only the trans zwitterion can be converted to the anion through a short proton wire. Therefore, an ultra-fast deactivation channel is available only for the trans zwitterion, and not for the fluorescent cis zwitterion. From the presence of the hydrogen bonding network in our force field trajectories, we do not obtain insights into the energetics of proton transfer. Studying these transfers along the identified pathways in asFP595, both in the ground and the excited state, is beyond the scope of the present work.


Chromophore protonation state controls photoswitching of the fluoroprotein asFP595.

Schäfer LV, Groenhof G, Boggio-Pasqua M, Robb MA, Grubmüller H - PLoS Comput. Biol. (2008)

Hydrogen bonding network in the chromophore cavity during force field simulations of zwitterionic chromophores.(A, C) Snapshots from MD simulations of Ztrans and Zcis, respectively. The blue dashed lines indicates the distance between the NH proton of MYG and Glu215, and the red dashed line that between the OH-group of Glu215 and the Nδ atom of His197. (B, D) Time-evolution of the two hydrogen bonds shown in (A) and (C) during representative force field MD simulations.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2274881&req=5

pcbi-1000034-g006: Hydrogen bonding network in the chromophore cavity during force field simulations of zwitterionic chromophores.(A, C) Snapshots from MD simulations of Ztrans and Zcis, respectively. The blue dashed lines indicates the distance between the NH proton of MYG and Glu215, and the red dashed line that between the OH-group of Glu215 and the Nδ atom of His197. (B, D) Time-evolution of the two hydrogen bonds shown in (A) and (C) during representative force field MD simulations.
Mentions: To identify possible ESPT pathways, we have carried out extended force field MD simulations of both Ztrans and Zcis and analyzed the relevant hydrogen bonds. Figure 6A shows that, during the simulation of Ztrans, two stable hydrogen bonds were formed between the protonated OH group of Glu215 and His197 as well as between the NH proton of MYG and Glu215. These two hydrogen bonds allow for a proton transfer from Ztrans to the rapidly deactivating Atrans. The OH proton of Glu215 could transfer to the Nδ atom of His197, with a simultaneous or subsequent transfer of the NH proton of the imidazolinone moiety to Glu215. In contrast, during the force field simulation of Zcis, the MYG-Glu215 hydrogen bond remained intact, whereas the Glu215-His197 hydrogen bond broke after about 1 ns (Figure 6B). This differential behavior of Ztrans and Zcis was confirmed by two additional independent MD simulations (data not shown). Based on these results, we assume that only the trans zwitterion can be converted to the anion through a short proton wire. Therefore, an ultra-fast deactivation channel is available only for the trans zwitterion, and not for the fluorescent cis zwitterion. From the presence of the hydrogen bonding network in our force field trajectories, we do not obtain insights into the energetics of proton transfer. Studying these transfers along the identified pathways in asFP595, both in the ground and the excited state, is beyond the scope of the present work.

Bottom Line: We have identified the tight coupling of trans-cis isomerization and proton transfers in photoswitchable proteins to be essential for their function and propose a detailed underlying mechanism, which provides a comprehensive picture that explains the available experimental data.The structural similarity between asFP595 and other fluoroproteins of interest for imaging suggests that this coupling is a quite general mechanism for photoswitchable proteins.These insights can guide the rational design and optimization of photoswitchable proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Theoretical and Computational Biophysics, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany.

ABSTRACT
Fluorescent proteins have been widely used as genetically encodable fusion tags for biological imaging. Recently, a new class of fluorescent proteins was discovered that can be reversibly light-switched between a fluorescent and a non-fluorescent state. Such proteins can not only provide nanoscale resolution in far-field fluorescence optical microscopy much below the diffraction limit, but also hold promise for other nanotechnological applications, such as optical data storage. To systematically exploit the potential of such photoswitchable proteins and to enable rational improvements to their properties requires a detailed understanding of the molecular switching mechanism, which is currently unknown. Here, we have studied the photoswitching mechanism of the reversibly switchable fluoroprotein asFP595 at the atomic level by multiconfigurational ab initio (CASSCF) calculations and QM/MM excited state molecular dynamics simulations with explicit surface hopping. Our simulations explain measured quantum yields and excited state lifetimes, and also predict the structures of the hitherto unknown intermediates and of the irreversibly fluorescent state. Further, we find that the proton distribution in the active site of the asFP595 controls the photochemical conversion pathways of the chromophore in the protein matrix. Accordingly, changes in the protonation state of the chromophore and some proximal amino acids lead to different photochemical states, which all turn out to be essential for the photoswitching mechanism. These photochemical states are (i) a neutral chromophore, which can trans-cis photoisomerize, (ii) an anionic chromophore, which rapidly undergoes radiationless decay after excitation, and (iii) a putative fluorescent zwitterionic chromophore. The overall stability of the different protonation states is controlled by the isomeric state of the chromophore. We finally propose that radiation-induced decarboxylation of the glutamic acid Glu215 blocks the proton transfer pathways that enable the deactivation of the zwitterionic chromophore and thus leads to irreversible fluorescence. We have identified the tight coupling of trans-cis isomerization and proton transfers in photoswitchable proteins to be essential for their function and propose a detailed underlying mechanism, which provides a comprehensive picture that explains the available experimental data. The structural similarity between asFP595 and other fluoroproteins of interest for imaging suggests that this coupling is a quite general mechanism for photoswitchable proteins. These insights can guide the rational design and optimization of photoswitchable proteins.

Show MeSH
Related in: MedlinePlus