Aggregation kinetics of the protein photoreceptor Vivid.

The aggregation of proteins is of importance in fields ranging from protein homeostasis to disease. The light-sensing protein Vivid (VVD) regulates responses to blue-light illumination in the filamentous fungus Neurospora crassa. Consisting of a single light‑oxygen-voltage domain, VVD is characterized by cycling between dark and lit states that correspond to formation and disruption of a photoadduct between the flavin cofactor and the apoprotein. Recently, in vitro assays have shown that VVD undergoes self-oxidative damage and aggregation resulting from excessive blue-light illumination. To explore the aggregation process of VVD, here we study the kinetics of aggregation and how it is influenced by environmental factors such as initial protein concentration, temperature, and light. We found that the aggregation kinetics of VVD is consistent with a second-order reaction model involving kinetic control, where thermal decay from lit-VVD to dark-VVD is necessary for aggregation to proceed. The height of the energy barrier separating the lit and dark VVD states is measured as (80 ± 2) kJ mol-1. Application of the kinetic model to the observed dependence of aggregation vs. temperature allowed us to further estimate the energy involved in the nucleation of dark-VVD, (257 ± 24) kJ mol-1. Finally, we show that VVD aggregation levels increase as the time of blue light exposure is augmented, suggesting possible mechanisms for protein damage. These results demonstrate how aggregation of a photoreceptor depends not only on environmental factors but on the intrinsic response of the protein to illumination.