Local Chain Dynamics of Intrinsically Disordered Sic1 Protein Inferred from Fluorescence Anisotropy Decay Measurements

Abstract

As part of the cell cycle of the budding yeast Saccharomyces cerevisiae, the cyclin-dependent kinase inhibitor Sic1 is an intrinsically disordered protein (IDP) that is degraded by the Cell division control protein 4 (Cdc4) substrate of the SCF ubiquitin ligase complex upon multi-site phosphorylation. More specifically, the binding affinity of Sic1 to the WD40 binding domain of Cdc4 increases nonlinearly with the number of phosphate groups added to the Sic1 sequence. Previous studies have shown that the overall compaction of Sic1 is not altered significantly upon phosphorylation and/or binding. In fact, the Sic1 protein remains disordered upon binding and it is thought to form a “fuzzy” complex with Cdc4. The internal chain dynamics of the disordered region of Sic1 were examined by fluorescence anisotropy decay (FAD) measurements using site-specific labelling with Alexa Fluor-488 at six different positions along the chain. Anisotropy decays for each labelled construct were fit with two rotational correlation times (∼0.3 ns and ∼2 ns), much faster than the tumbling rate of the whole protein. Conversely, these data provide a map of the dye/linker rotational freedom and the intrinsic backbone torsional mobility of separate regions in the Sic1 sequence. Differential quenching as measured by the fluorescence lifetime points to local/distant contacts between some of the labelling sites and the only aromatic residue in Sic1 (Y14). Additionally, the backbone segmental dynamics are due to dihedral angle fluctuations, and they were compared with a Ramachandran plot obtained from the (p)Sic1 conformations available in the ENSEMBLE database. Going forward, chain dynamics data obtained from fluorescence anisotropy decays can be incorporated as an additional (local) constraint in ENSEMBLE to complement existing constraints from NMR (local) and SAXS (global).