STRUCTURAL CHARACTERIZATION OF NATURAL AND DESIGNED PHOTOSENSORS OF THE LIGHT

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Perception and integration of environmental stimuli are elemental for all living organisms and are frequently mediated by members of the versatile Per-Arnt-Sim (PAS) family which occur in all kingdoms of life. A subclass of the PAS family are the light-oxygen-voltage (LOV) proteins which bind flavin mononucleotide (FMN) as a cofactor. Upon blue light illumination LOV photosensors form a covalent bond between a cysteine residue and FMN. Our research aims at understanding and controlling signal generation and propagation in LOV proteins. Two main lines of research are pursued. First, we will study light signaling in natural LOV proteins. Our research focuses on how protein structure and dynamics are influenced by light and how the initial signal is relayed to downstream effectors. High-resolution structures of LOV domains and their conjugate effectors will provide mechanistic insight into these processes. We also plan to obtain structural information on the photoactivated state of these proteins either by freeze trapping techniques or time-resolved X-ray crystallography. The latter also allows to access transiently populated intermediates during the photocycle. In a second approach artificial proteins based on LOV domains will be designed using molecular biology and in vitro evolution techniques. By covalently linking effector proteins to LOV domains, we want to place their function under light control. Structural information both on stable and transiently populated states will be essential for elucidating the molecular mechanism of these proteins. The successful design of novel light-regulated proteins would improve our understanding of the signaling process and could be used in biotechnological and other applications. Comparison to results obtained on natural proteins highlights similarities and differences which should improve our knowledge about signaling in LOV domains. Our research could also prove relevant for signal transduction in the larger PAS family.