1.
Light-inducible system for tunable protein expression in Neurospora crassa.
Abstract:
Filamentous fungi are important model systems for understanding eukaryotic cellular processes, including the study of protein expression. A salient feature of fungi is the ability of the protein-processing machinery to perform all of the extensive posttranslational modifications needed in the complex world of eukaryotic organisms, making them great hosts for production of eukaryotic proteins. In the model organism Neurospora crassa, several regulatable promoters have been used for heterologous gene expression but all suffer from leaky expression absent stimuli or an inability to induce protein expression at levels greater than those seen in vivo. To increase and better control in vivo protein expression in Neurospora, we have harnessed the light-induced vvd promoter. vvd promoter-driven mRNA expression is dependent upon light, shows a graded response, and is rapidly shut off when returned to the dark. The vvd promoter is a highly tunable and regulatable system, which could be a useful instrument for those interested in efficient and controllable gene expression.
2.
Structure of a light-activated LOV protein dimer that regulates transcription.
Abstract:
Light, oxygen, or voltage (LOV) protein domains are present in many signaling proteins in bacteria, archaea, protists, plants, and fungi. The LOV protein VIVID (VVD) of the filamentous fungus Neurospora crassa enables the organism to adapt to constant or increasing amounts of light and facilitates proper entrainment of circadian rhythms. Here, we determined the crystal structure of the fully light-adapted VVD dimer and reveal the mechanism by which light-driven conformational change alters the oligomeric state of the protein. Light-induced formation of a cysteinyl-flavin adduct generated a new hydrogen bond network that released the amino (N) terminus from the protein core and restructured an acceptor pocket for binding of the N terminus on the opposite subunit of the dimer. Substitution of residues critical for the switch between the monomeric and the dimeric states of the protein had profound effects on light adaptation in Neurospora. The mechanism of dimerization of VVD provides molecular details that explain how members of a large family of photoreceptors convert light responses to alterations in protein-protein interactions.
