Electrostatics and Intrinsic Disorder: A Single-Molecule Study of the Sic1 Protein

Abstract

Intrinsically disordered proteins (IDPs) play critical yet often poorly understood roles in a variety of cellular processes. Despite an apparent antagonism between structural disorder and protein recognition, the large number of IDPs involved in protein regulation suggests that they could in fact provide advantages in recognition over well-folded proteins. Sic1 is an IDP inhibitor of a cyclin dependent kinase (CDK) in yeast, which interacts with a single site on its acceptor Cdc4 only upon phosphorylation of its multiple dispersed CDK sites. The multiple phosphorylation events can in principle be the basis for ultrasensitivity in protein-protein binding, however its molecular basis remains elusive1.We performed a systematic study of the Sic1's fluctuating conformations in different salt and denaturant concentrations using single-molecule Forster energy transfer (smFRET) and fluorescence correlation spectroscopy (FCS). smFRET data suggests that Sic1 protein is comprised of a continuum of conformers with varying end-end distances. Denaturant titration measurements suggest that these conformers are characterized by non-cooperative interactions. From FCS experiments, the exchange between Sic1 conformational states was found to occur on both ultrafast (10-100 ns timescale) and slow (10-100 ms) timescale. Burst smFRET experiments show that Sic1’s end-end distances do not vary significantly upon addition of salt, which suggests that charge-shielding by salt may only affect the structure locally, around charged amino acids. Our single-molecule data resolves conformational heterogeneity and dynamics in a model IDP protein and represent the first step towards the validation of the polyelectrostatic model of the Sic1-Cdc4 interaction2.(1) Nash, P., Tang, X., Orlicky, S., Chen, Q., Gertler, F. B., Mendenhall, M. D., Sicheri, F., Pawson, T., Tyers, M. Nature 2001, 414, 514.(2) Borg, M., Mittag, T., Pawson, T., Tyers, M., Forman-Kay, J. D., Chan, H. S. Proc. Natl. Acad. Sci. USA 2007, 104, 9650.