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 longrange 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.

Choosing the right fluorophore for single-molecule fluorescence studies in a lipid environment

“…Depending on the window required for intramolecular distance measurements and on the instrument capabilities, blue-green, green-red or blue-red fluorophore pairs are commonly used [7,33,34]. To meet the requirement for a short Förster radius, a blue-red fluorophore pair is typically preferred because of the small spectral overlap between the donor and the acceptor.…”

“…Fluorescence decay measurements were performed on a custombuilt multiparameter epi-fluorescence microscope [32][33][34]. Pulsed laser excitation at 480 nm and 527 nm was provided by frequency-doubling the output of a tunable femtosecond oscillator (Tsunami HP, Spectra Physics).…”

The contribution of intrinsically disordered regions to protein function, cellular complexity, and human disease

“…An important aspect by which IDRs contribute to protein function is by adopting a defined conformation when binding a specific interaction partner [6,9,32,34,53,71–74]. Although a large fraction of the polypeptide adopts a defined structure upon complex formation, distinct segments can still remain disordered.…”

Characterization of Fluorescein Arsenical Hairpin (FlAsH) as a Probe for Single-Molecule Fluorescence Spectroscopy

“…A multiparameter fluorescence (MPF) confocal microscope 50 with alternating-laser excitation (ALEX) 39 was used to characterize the dually labelled peptide, FlAsH-FCMybbR-CoA-AF647, at the level of single- molecule bursts of fluorescence (Fig. 6a).…”