title: The effect of intrachain electrostatic repulsion on conformational disorder and dynamics of the Sic1 protein.
abstract: The yeast cyclin-dependent kinase inhibitor Sic1 is a disordered protein that, upon multisite phosphorylation, forms a dynamic complex with the Cdc4 subunit of an SCF ubiquitin ligase. To understand the multisite phosphorylation dependence of the Sic1:Cdc4 interaction, which ultimately leads to a sharp cell cycle transition, the conformational properties of the disordered Sic1 N-terminal targeting region were studied using single-molecule fluorescence spectroscopy. Multiple conformational populations with different sensitivities to charge screening were identified by performing experiments in nondenaturing salts and ionic denaturants. Both the end-to-end distance and the hydrodynamic radius decrease monotonically with increasing the salt concentration, and a rollover of the chain dimensions in high denaturant conditions is observed. The data were fit to the polyelectrolyte binding-screening model, yielding parameters such as the excluded volume of the uncharged chain and the binding constant to denaturant. An overall scaling factor of ∼1.2 was needed for fitting the data, which implies that Sic1 cannot be approximated by a random Gaussian chain. Fluorescence correlation spectroscopy reveals Sic1 structure fluctuations occurring on both fast (10-100 ns) and slow (∼10 ms) time scales, with the fast phase absent in low salt solutions. The results of this study provide direct evidence that long-range intrachain electrostatic repulsions are a significant factor for the conformational landscape of Sic1, and support the role of electrostatics in determining the overall shape and hydrodynamic properties of intrinsically disordered proteins.
authors.affiliation: Department of Chemical and Physical Sciences, University of Toronto Mississauga , Mississauga, Ontario L5L 1C6, Canada.
authors.forename: Baoxu
authors.initials: B
authors.lastname: Liu
authors.forename: Darius
authors.initials: D
authors.lastname: Chia
authors.forename: Veronika
authors.initials: V
authors.lastname: Csizmok
authors.forename: Patrick
authors.initials: P
authors.lastname: Farber
authors.forename: Julie D
authors.initials: JD
authors.lastname: Forman-Kay
authors.forename: Claudiu C
authors.initials: CC
authors.lastname: Gradinaru
mesh_terms: D050756:Cyclin-Dependent Kinase Inhibitor Proteins / Q000737:chemistry*; D057446:Hydrodynamics; D011487:Protein Conformation; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D055672:Static Electricity
chemical_list: D050756:Cyclin-Dependent Kinase Inhibitor Proteins; C086926:SIC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins