X-ray diffraction analysis of pressure-induced structural changes in the Aequorea yellow fluorescent protein Citrine reveals the structural basis for the continuous fluorescence peak shift from yellow to green that is observed on pressurization. This fluorescence peak shift is caused by a reorientation of the two elements of the Citrine chromophore. This study describes the structural linkages in Citrine that are responsible for the local reorientation of the chromophore. The deformation of the Citrine chromophore is actuated by the differential motion of two clusters of atoms that compose the β-barrel scaffold of the molecule, resulting in a slight bending of the β-barrel. The high-pressure structures also show a perturbation of the hydrogen bonding network that stabilizes the excited state of the Citrine chromophore. The perturbation of this network is implicated in the reduction of fluorescence intensity of Citrine. The blue-shift of the Citrine fluorescence spectrum resulting from the bending of the β-barrel provides structural insight into the transient blue-shifting of isolated yellow fluorescent protein molecules under ambient conditions and suggests mechanisms to alter the time-dependent behavior of Citrine under ambient conditions.
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