Structure-function analyses of mCRY-mPER2 and mCRY-mBMAL1 complexes

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Most organisms exhibit daily cycles of physiology, metabolism and behaviour, so called circadian rhythms, which are generated by circadian clocks. Disruption of the normal circadian (~ 24 h) cycle e.g. due to mutations, jet-lag or shift-work severely compromises human physiology, as revealed, for example, by increased incidences of cancer and metabolic syndrome in shift workers. The ~24 h period is generated by gene-regulatory negative feedback loops with a spatio-temporally regulated interplay of synthesis, posttranslational modifications, interactions and degradation of clock proteins. Here we propose to study two essential protein-protein complexes of the mammalian circadian clock, the Cryptochrome1/2-mPERIOD2 (mCRY-mPER2)- and the Cryptochrome1/2-mBMAL1 (mCRY-mBMAL1) complex, at multiple levels ranging from high-resolution structural insights to circadian phenotypes in vivo. Methods will include X-ray crystallography, protein interaction studies (e.g. analytical gel filtration, isothermal titration calorimetry, fluorescence polarization, pull-down, native polyacrylamide gels) as well as functional and phenotypic analyses in live cells (e.g. luciferase complementation, fluorescence-two-hybrid, co-immunoprecipitation, FRET, FRAP, cotransactivation, cellular localization and stability, circadian oscillations). Our studies will provide important mechanistic insights into the regulation of mCRY and mPER2 protein stability and cellular localization and into the mCRY-dependent transcriptional repression of the mBMAL/mCLOCK complex. These molecular processes are essential for maintaining the 24 h period of the circadian clock and of clock regulated human behaviour and physiology. Furthermore, our structures will advance the design of CRY chemical probes and of novel therapeutic metabolic regulators.