Disordered Motifs and Domains in Cell Control - October 11-15, 2014

Disordered Motifs and Domains in Cell Control

Poster Session I

6-POS Board 6 The Effect of Intrachain Electrostatic Repulsion on the Conformational Disorder and Dynamics of the Sic1 Protein: A Single-molecule Study Claudiu Gradinaru 1 , Baoxu Liu 1 , Gregory-Neal Gomes 1 , Patrick Farber 2 , Veronika Csizmok 2 , Julie Forman-Kay 2 . 1 University of Toronto, Toronto, ON, Canada, 2 Hospital for Sick Children, Toronto, ON, Canada. In yeast, the cyclin-dependent kinase inhibitor Sic1 is a disordered protein that, upon multi-site phosphorylation, forms a dynamic complex with the Cdc4 subunit of an SCF ubiquitin ligase. To understand the multi-site 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 smFRET and FCS experiments in non-denaturing 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 was fitted 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 could imply 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 (~10ms) time scales, with the fast phase absent in low salt solutions. Our data 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.

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