Tracking the structural dynamics of photoreceptor proteins: LOV2 using Time resolved X-ray Scattering Techniques and TG studies

Abstract

Phototropins belongs to the class of blue light activated protein kinases which are essential for phototropism in plants. Phototropin has two chromophoric domains named light-oxygen-voltage- sensing LOV1 and LOV2 in the N-terminal and a serine/threonine (Ser/Thr) protein kinase motif in the C-terminal connected by Jα helix. These domains bind to flavin chromophore and form a FMN-Cys photoadduct as part of light sensing process and make the Jα helix to unfold followed by activation of kinase domain. It remains unclear so far that how the signal is transmitted to the kinase domain from the LOV2 core at atomic level.To study the conformational changes of protein in the native aqueous environment is a challenging task.In this work, we have employed picosecond X-ray solution scattering and novel structural analysis to track the details of the structural dynamics of I427V mutant of LOV2 over time range from 10 us to 10 sec, since it has quick recovery time scale of about 5 sec that is suitable for the time resolved X-ray scattering techniques in order to study the folding dynamics. Moreover, we will track the intermediates that form during the reaction pathway of wild type LOV2 at molecular level. Also, we are trying to design novel photoreceptors using LOV2 domain and to study the signal propagation from the sensory domain to the DNA binding domain (RAC1) by using Transient grating techniques, as it has wide application in optogenetic studies. Also bacterial photoreceptor proteins like BlrB and AppA was also cloned in pET15b vector and expressed well in BL21DE3 cell line for TR-WAXS and TG studies.