Global Dimensions Are Decoupled From Electrostatics In The Intrinsically Disordered Protein Sic1

Sic1 is a disordered cyclin-dependent kinase inhibitor which must be phosphorylated on at least six sites (termed Cdc4 phosphodegrons, CPDs) to allow its recognition by the WD40 binding domain of Cdc4. The highly-cooperative switch-like dependence on the number of phosphorylated sites on Sic1 cannot be accounted for by traditional thermodynamic models of cooperativity. Further experimental attention is necessary to determine the physico-chemical basis of its highly cooperative binding. We used single molecule fluorescence techniques to study the dimensions and dynamics of Sic1's N-terminal targeting region (residues 1-90, henceforth Sic1) and phosphorylated Sic1 (pSic1). A quantitative relationship between sequence properties and ensemble properties is a prerequisite for understanding IDP phosphorylation and its role in highly cooperative binding. Theories which seek a quantitative relationship between the primary amino-acid sequence of an IDP and its conformational properties predominantly consider sequences of IDPs that are not phosphorylated and which are deficient in proline residues. We find that Sic1's global dimensions, such as the radius of gyration, are decoupled from electrostatic determinants (phosphorylation, charge screening). We analyze the resulting conformational properties using polymer theory sequence-ensemble relationships. Furthermore, a quantitative relationship between sequence properties and ensemble properties requires the accurate determination of ensemble dimensions. We analyze our single molecule FRET data (smFRET) using homopolymer models, and by comparison with NMR- and SAXS- restrained ENSEMBLES. We find that homopolymer models fail to infer accurate radius of gyration in the presence of significant transient structure, though inferences of root-mean-squared end-to-end distance appear to be accurate. The ENSEMBLES are directly compared to the smFRET data by comparing with the calculated mean FRET efficiency. The consistency shown will allow future ENSEMBLE calculations to use smFRET as a restraint.