Intrinsic stiffness and -solvent regime in intrinsically disordered proteins: Implications for liquid-liquid phase separation.

Liquid-liquid phase separation (LLPS) exhibited by intrinsically disordered proteins (IDPs) depends on the solvation state around the -regime, which separates good from poor solvent. Experimentally, the -solvent regime of the finite length () IDPs, as probed by small angle X-ray scattering (SAXS) and single molecular fluorescence resonance energy transfer (smFRET), is in disagreement. Using computer simulations of a coarse-grained IDP model, we address the effect of chain length on the -regime of IDPs with polar side chains (polyglutamine) and hydrophobic side chains (polyleucine) subject to varying concentrations of cosolvents , urea (denaturant) or trimethylamine N-oxide (protective osmolyte) in water. Due to their intrinsic stiffness, these IDPs are always expanded on short-length scales, independent of the solvent quality. As a result, for short IDP sequences ( to 25 residues), their propensity to exhibit LLPS cannot be inferred from single-chain properties. Further, for finite-size IDPs, the cosolvent concentration to attain the -regime ( ) extracted from the structure factor emulating SAXS and pair distances mimicking smFRET differs. They converge to the same cosolvent concentration only at large , indicating that finite size corrections vary for different IDP properties. We show that the radius of gyration ( ) of the IDPs in the -solvent regime satisfies the scaling relation , which can be exploited to accurately extract ( ). We demonstrate the importance of finite size aspects originating from the chain stiffness and thermal blob size in analyzing IDP properties to identify the -solvent regime.