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    Structural events in the photocycle of green fluorescent protein

    van Thor, J.J. and Zanetti, Giulia and Ronayne, K.L. and Towrie, M. (2005) Structural events in the photocycle of green fluorescent protein. The Journal of Physical Chemistry B 109 (33), pp. 16099-16108. ISSN 1520-6106.

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    Abstract

    Picosecond time-resolved mid-infrared absorption changes of the wild type green fluorescent protein from Aequorea victoria are reported on structural events during the photocycle. Concomitant with rapid H/D transfer following excitation of the neutral A state at 400 nm, a transient signal at 1721/1711 cm-1 (H/D) developed belonging to protonated glutamate 222, which was definitively assigned using the E222D mutant from the altered proton-transfer kinetics to aspartate in addition to the altered band position and intensity in the spectra. A transient at 1697 cm-1, assigned to a structural perturbation of glutamine 69, had a H/D kinetic isotope effect of >32, showing the conformational dynamics to be sensitive to the active site H/D vibrations. The kinetic data up to 2 ns after excitation in the 1250−1800 cm-1 region in D2O provided 10 and 75 ps time constants for the excited-state deuteron transfer and the associated A1* − A1 and A2* − A2 difference spectra and showed the radiative intermediate I* state vibrations and the transient accumulation of the long-lived ground-state intermediate I2. Assignments of chromophore modes for the A1, A2, and I2 ground states are proposed on the basis of published model compound studies1,2 (Esposito, A. P.; Schellenberg, P.; Parson, W. W.; Reid, P. J. J. Mol. Struct. 2001, 569, 25 and He, X.; Bell, A. F.; Tonge, P. J. J. Phys. Chem. B 2002, 106, 6056). Tentative assignments for the singlet-state intermediates A1*, A2*, and I* are discussed. An unexpected and unassigned band that may be a CC chromophore vibration was observed in the ground state (1665 cm-1) as well as in all photocycle intermediates. Optical dumping of the transient I* population was achieved using an additional 532 nm pulse and the directly obtained I2 − I* difference spectrum was highly similar to the I2 − I* photocycle spectrum. The pump−dump−probe spectrum differed from the pump−probe photocycle difference spectrum with respect to the intensity of the phenol 1 mode at 1578 cm-1, suggesting stronger delocalization of the negative charge onto the phenolic ring of the anionic chromophore in the dumped I2 state. Indication for structural heterogeneity of the chromophore, Glu 222, and the chromophore environment was found in the two parallel proton-transfer reactions and their distinct associated ground- and intermediate-state vibrations. Vibrational spectral markers at 1695 cm-1 assigned to Gln 69, at 1631 cm-1 belonging to a CC mode, and at 1354 cm-1 belonging to a phenolate vibration further indicated the I2 and I* states to be unrelaxed.

    Metadata

    Item Type: Article
    School: Birkbeck Schools and Departments > School of Science > Biological Sciences
    Depositing User: Sarah Hall
    Date Deposited: 20 May 2019 11:28
    Last Modified: 20 May 2019 11:28
    URI: http://eprints.bbk.ac.uk/id/eprint/27574

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