Chemical perturbation of the conformational change in the orange carotenoid protein

Fugui Xiaoa, Rob B.M. Koehorsta,b, Herbert van Amerongena,b
aLaboratory of Biophysics, Wageningen University, P.O. Box 8128, 6700 ET Wageningen, The Netherlands; bMicroSpectroscopy Centre, Wageningen University, P.O. Box 8128, 6700 ET Wageningen, The Netherlands

Photosynthetic organisms exploit different mechanisms to prevent oxidative damage under high-light conditions. In cyanobacteria, the orange carotenoid protein (OCP) is responsible for quenching a large part of the energy absorbed by the light-harvesting complexes. Upon strong illumination the protein undergoes a conformational change from the inactive orange form (OCPO), to the active red conformation (OCPR), which is capable of quenching excitation energy. Previous studies have shown that destabilizing ions such as thiocyanate can induce the red conformation in the absence of illumination, whereas stabilizing ions such as phosphate can reduce the transition to the red conformation in the presence of illumination.

The mechanism of the OCP conformational transformation in ions is still not clear, and the study of it can provide clue on the conformational change under blue green light.  Here, we test whether the widely used destabilizing chemicals urea and guanidine hydrochloride (GdnHCl) have a similar effect on OCP as thiocyanate, and whether the widely used stabilizing chemical Trimethylamine N-oxide (TMAO) has a similar effect on OCP as phosphate. On the one hand, no red form accumulates in 2M TMAO in the presence of intense light, suggesting that a conformational change occurs in the protein part of OCP upon absorbing blue green light. On the other hand, although 1.5M sodium thiocyanate is able to induce OCPR in the dark, no red form accumulates in 4M urea in the dark.  In contrast to the low efficiency of urea, 49% of OCP in its red form in 1M GdnHCl, and 90% in 2M GdnHCl. As a protein denaturant, GdnHCl is generally 2-2.5-fold more effective than urea, but GdnHCl is at least 4 times more effective in inducing OCPR than urea. Furthermore, it is found that in 0.5M GdnHCl the transition from the red to the orange form takes far longer than in 4M urea (27 vs. 5 minutes).

The difference in behavior is ascribed to the ionic properties of GdnHCl, which can perturb salt bridges on the protein surface.  This was further tested by comparing the switching kinetics in the absence and presence of 0.5M NaCl. In the latter case the kinetics slowed down from 1 to 7 minutes, suggesting that salt bridges are indeed involved in the conformational change. This conclusion was further confirmed by the observation that OCP in the dark goes to the red form at pH < 2.2 or > 9.5.