Two-step structural changes in orange carotenoid protein photoactivation revealed by time-resolved Fourier transform infrared spectroscopy

Abstract

The orange carotenoid protein (OCP), which is essential in cyanobacterial photoprotection, is the first photoactive protein containing a carotenoid as an active chromophore. Static and time-resolved Fourier transform infrared (FTIR) difference spectroscopy under continuous illumination at different temperatures was applied to investigate its photoactivation mechanism. Here, we demonstrate that in the OCP, the photo-induced conformational change involves at least two different steps, both in the second timescale at 277 K. Each step involves partial reorganization of α-helix domains. At early illumination times, the disappearance of a nonsolvent-exposed α-helix (negative 1651 cm–1 band) is observed. At longer times, a 1644 cm–1 negative band starts to bleach, showing the disappearance of a solvent-exposed α-helix, either the N-terminal extension and/or the C-terminal tail. A kinetic analysis clearly shows that these two events are asynchronous. Minor modifications in the overall FTIR difference spectra confirm that the global protein conformational change consists of—at least—two asynchronous contributions. Comparison of spectra recorded in H2O and D2O suggests that internal water molecules may contribute to the photoactivation mechanism.