Prion-like domains (PLDs) can drive liquid-liquid phase separation (LLPS) in cells. Using an integrative biophysical approach that includes nuclear magnetic resonance spectroscopy, small-angle x-ray scattering, and multiscale simulations, we have uncovered sequence features that determine the overall phase behavior of PLDs. We show that the numbers (valence) of aromatic residues in PLDs determine the extent of temperature-dependent compaction of individual molecules in dilute solutions. The valence of aromatic residues also determines full binodals that quantify concentrations of PLDs within coexisting dilute and dense phases as a function of temperature. We also show that uniform patterning of aromatic residues is a sequence feature that promotes LLPS while inhibiting aggregation. Our findings lead to the development of a numerical stickers-and-spacers model that enables predictions of full binodals of PLDs from their sequences.

Prion-like low complexity domains (PLCDs) have distinctive sequence grammars that determine their driving forces for phase separation. Here, we uncover the physicochemical underpinnings of how evolutionarily conserved compositional biases influence the phase behavior of PLCDs. We interpret our results in the context of the stickers-and-spacers model for phase separation of associative polymers. We find that tyrosine is a stronger sticker than phenylalanine whereas arginine is a context dependent auxiliary sticker. In contrast, lysine weakens sticker-sticker interactions. Increasing the net charge per residue destabilizes phase separation while also weakening the strong coupling between single-chain contraction in dilute phases and multi-chain interactions that give rise to phase separation. Finally, glycine and serine residues act as non-equivalent spacers, thus making glycine vs. serine contents an important determinant of the driving forces for phase separation. The totality of our results leads to a set of rules that enable comparative estimates of composition-specific driving forces for PLCD phase separation.

Phase separation of intrinsically disordered prion-like low-complexity domains (PLCDs) derived from RNA-binding proteins enable the formation of biomolecular condensates in cells. PLCDs have distinct amino acid compositions, and here we decipher the physicochemical impact of conserved compositional biases on the driving forces for phase separation. We find that tyrosine residues make for stronger drivers of phase separation than phenylalanine. Depending on their sequence contexts, arginine residues enhance or weaken phase separation, whereas lysine residues weaken cohesive interactions within PLCDs. Increased net charge per residue (NCPR) weakens the driving forces for phase separation of PLCDs and this effect can be modeled quantitatively. The effects of NCPR also weaken known correlations between the dimensions of single chains in dilute solution and the driving forces for phase separation. We build on experimental data to develop a coarse-grained model for accurate simulations of phase separation that yield novel insights regarding PLCD phase behavior.

Proteins are marginally stable molecules that fluctuate between folded and unfolded states. Here, we provide a high-resolution description of unfolded states under refolding conditions for the N-terminal domain of the L9 protein (NTL9). We use a combination of time-resolved Förster resonance energy transfer (FRET) based on multiple pairs of minimally perturbing labels, time-resolved small-angle X-ray scattering (SAXS), all-atom simulations, and polymer theory. Upon dilution from high denaturant, the unfolded state undergoes rapid contraction. Although this contraction occurs before the folding transition, the unfolded state remains considerably more expanded than the folded state and accommodates a range of local and nonlocal contacts, including secondary structures and native and nonnative interactions. Paradoxically, despite discernible sequence-specific conformational preferences, the ensemble-averaged properties of unfolded states are consistent with those of canonical random coils, namely polymers in indifferent (theta) solvents. These findings are concordant with theoretical predictions based on coarse-grained models and inferences drawn from single-molecule experiments regarding the sequence-specific scaling behavior of unfolded proteins under folding conditions.

In vitro, replacing KCl by potassium glutamate (KGlu), the E. coli cytoplasmic salt and osmolyte, stabilizes folded proteins and protein-nucleic acid complexes. To understand the chemical basis for these effects and rank Glu− in the Hofmeister anion series for protein unfolding, we quantify and interpret the strong stabilizing effect of KGlu on the ribosomal protein domain NTL9, relative to other stabilizers (KCl, KF, K2SO4) and destabilizers (GuHCl, GuHSCN). GuHSCN titrations at 20 °C, performed as a function of concentration of KGlu or other salt and monitored by NTL9-fluorescence, are analyzed to obtain r-values quantifying the Hofmeister salt concentration (m3)-dependence of the unfolding equilibrium constant Kobs (r-value = −dlnKobs/dm3 = (1/RT) dΔG°obs/dm3 = m-value/RT). r-Values for both stabilizing K+ salts and destabilizing GuH+ salts are compared with predictions from model-compound data. For two-salt mixtures, we find that contributions of stabilizing and destabilizing salts to observed r-values are additive and independent. At 20 °C, we determine a KGlu r-value of 3.22 m−1, and K2SO4, KF, KCl, GuHCl and GuHSCN r-values of 5.38, 1.05, 0.64, −1.38 and −3.00 m−1 respectively. The KGlu r-value represents a 25-fold (1.9 kcal) stabilization per molal KGlu added. KGlu is much more stabilizing than KF, and the stabilizing effect of KGlu is larger in magnitude than the destabilizing effect of GuHSCN. Interpretation of the data reveals good agreement between predicted and observed relative r-values, and indicates the presence of significant residual structure in GuHSCN-unfolded NTL9 at 20 °C.