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Chromophore protonation state controls photoswitching of the fluoroprotein asFP595.

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.

SUBMITTER: Schafer LV

PROVIDER: S-EPMC2274881 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Acid-sensing ion channels (ASICs) are neuronal Na(+)-conducting channels activated by extracellular acidification. ASICs are involved in pain sensation, expression of fear, and neurodegeneration after ischemic stroke. Functional ASICs are composed of three identical or homologous subunits, whose extracellular part has a handlike structure. Currently, it is unclear how protonation of residues in extracellular domains controls ASIC activity. Knowledge of these mechanisms would allow a rational development of drugs acting on ASICs. Protonation may induce conformational changes that control the position of the channel gate. We used voltage-clamp fluorometry with fluorophores attached to residues in different domains of ASIC1a to detect conformational changes. Comparison of the timing of fluorescence and current signals identified residues involved in movements that preceded desensitization and may therefore be associated with channel opening or early steps leading to desensitization. Other residues participated in movements intimately linked to desensitization and recovery from desensitization. Fluorescence signals of all mutants were detected at more alkaline pH than ionic currents. Their midpoint of pH dependence was close to that of steady-state desensitization, whereas the steepness of the pH fluorescence relationship was closer to that of current activation. A sequence of movements was observed upon acidification, and its backward movements during recovery from desensitization occurred in the reverse order, indicating that the individual steps are interdependent. Furthermore, the fluorescence signal of some labeled residues in the finger domain was strongly quenched by a Trp residue in the neighboring β-ball domain. Upon channel activation, their fluorescence intensity increased, indicating that the finger moved away from the β ball. This extensive analysis of activity-dependent conformational changes in ASICs sheds new light on the mechanisms by which protonation controls ASIC activity.

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Chromophore protonation state controls photoswitching of the fluoroprotein asFP595.

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