Crossover in aromatic amino acid interaction strength between tyrosine and phenylalanine in biomolecular condensates

For decades, cellular organization was primarily understood through the lens of membrane-bound compartmentalization. However, it has become increasingly clear that membrane-less organelles —such as stress granules, nuclear speckles, and Cajal bodies— are also widespread within cells (Banani et al., 2017). At least in some cases, these organelles form by phase separation of their components (Shin and Brangwynne, 2017), primarily disordered regions of proteins and nucleic acids, which partition into dense and dilute phases. The condensates thus formed retain properties of liquids, like fusion, dripping, and wetting (Brangwynne et al., 2009). This phenomenon has generated significant interest in the physical mechanisms governing the phase behaviour of biomolecular mixtures (Choi et al., 2020).

Many proteins undergoing phase separation share common characteristics, including deviations from typical compositions of folding proteins and simple sequences with multiple amino acids repeats. Specifically, in the sequences of proteins that experience upper critical solution temperature (UCST) transitions, like the low-complexity regions of FUS, hnRNPA1, Ddx4, LAF-1, or atGRP7, we observe long stretches rich in polar residues interspersed with aromatics or positively charged residues (Martin and Mittag, 2018). Borrowing language from the theory of associative polymers (Semenov and Rubinstein, 1998), these units of sequence have been termed ‘stickers,’ in the case of aromatics and positively charged residues, and ‘spacers,’ for the polar residue repeats (Martin et al., 2020; Bremer et al., 2022; Mittag and Pappu, 2022). Spacers act as linkers that lend flexibility to the polypeptide mesh in the protein-dense phase. In contrast, stickers play a key role in determining both the single-chain properties of the polymer and the phase behaviour of the condensate through interactions involving their aromatic or charged groups (Wang et al., 2018). Recent experiments have quantified the influence of different types of stickers on the polymer properties and phase behaviour (Bremer et al., 2022). This raises a crucial question: what is the fundamental origin of the relative strengths of different stickers?

This matter has recently been investigated in the context of the cationic amino acids lysine (Lys) and arginine (Arg) (Wang et al., 2018; Das et al., 2020; Schuster et al., 2020; Fisher and Elbaum-Garfinkle, 2020; Greig et al., 2020; Paloni et al., 2021; Hong et al., 2022). Despite having the same net charge, these residues turn out not to be interchangeable. Mutagenesis experiments on LAF-1 have shown that substituting Arg with Lys completely suppresses phase separation (Schuster et al., 2020). This distinction is functionally relevant, as Arg to Lys substitutions affect speckle formation (Greig et al., 2020). Additionally, experiments on the intrinsically disordered region (IDR) of Ddx4 indicate that phase separation is favoured by Arg relative to Lys (Brady et al., 2017; Das et al., 2020; Schuster et al., 2020). Das and co-workers attempted to explain arginine’s greater propensity to phase separate in Ddx4 variants using coarse-grained simulations with two different energy functions (Das et al., 2020). The model was first parametrized using a hydrophobicity scale, aimed to capture the ‘stickiness’ of different amino acids (Dignon et al., 2018), but this did not recapitulate the correct rank order in the stability of the simulated condensates (Das et al., 2020). By replacing the hydrophobicity scale with interaction energies from amino acid contact matrices —derived from a statistical analysis of the PDB (Dignon et al., 2018; Miyazawa and Jernigan, 1996; Kim and Hummer, 2008)— they recovered the correct trends (Das et al., 2020). A key to the greater propensity to phase separate in the case of Arg may derive from the pseudo-aromaticity of this residue, which results in a greater stabilization relative to the more purely cationic character of Lys (Gobbi and Frenking, 1993; Wang et al., 2018; Hong et al., 2022).

Here, we focus on the distinct roles of the main aromatic residues acting as stickers —tyrosine (Tyr) and phenylalanine (Phe)— excluding tryptophan due to its much lower abundance (Maraldo et al., 2024). Given that they only differ in a hydroxyl group, one could expect Tyr and Phe to be equally relevant to phase separation, especially considering the finding from a double-mutant cycle that Tyr-Tyr and Phe-Phe pairs make nearly identical contributions to protein stability (Serrano et al., 1991). However, experimental evidence on various proteins suggests that Tyr is a stronger driver of condensation (Lin et al., 2017; Wang et al., 2018; Schuster et al., 2020; Bremer et al., 2022). This is demonstrated by the reduced propensity to phase separate of Tyr-to-Phe mutants of FUS and LAF-1 (Lin et al., 2017; Wang et al., 2018; Schuster et al., 2020) and the opposite effect in Phe-to-Tyr mutants of hnRNPA1 (Wang et al., 2018; Bremer et al., 2022). Understanding the origin of the different sticker strengths of Phe and Tyr are important due to their functional relevance. In a large sample of hnRNPA1 variants, the fractions of Phe and Tyr have been found to co-vary, suggesting that evolutionary control of composition fine-tunes the properties of condensates (Bremer et al., 2022).

The molecular properties of Phe and Tyr may give important insights about their distinct behaviour as stickers in condensates. Hydrophobicity scales typically rank Phe as the most hydrophobic residue (Kyte and Doolittle, 1982; Tesei et al., 2021), consistent with the greater hydration free energy of Tyr relative to Phe (Wolfenden et al., 1981; Chang et al., 2007) (see Figure 1A–B). As in the case of Arg and Lys, using hydrophobicity as a proxy for interaction energy in bead simulation models proved insufficient to explain the relative strengths of Phe and Tyr as stickers (Das et al., 2020). However, in this case, statistical contact matrices also could not capture the correct order of stickiness. In the Miyazawa and Jernigan statistical potential, Tyr-Tyr contacts are weaker than Phe-Phe (with energies of –4.17 and –7.26 in RT units, respectively; see Figure 1C; Miyazawa and Jernigan, 1996). We note that a different hydrophobicity scale based on peptides undergoing inverse temperature transitions —i.e., the Urry scale (Urry et al., 1992), where Tyr is more hydrophobic— can account for the correct rank order in saturation concentration (Regy et al., 2021). On the other hand, the potentials of mean force between amino acids calculated with atomistic force fields have a deeper free energy well for the Tyr-Tyr pair than for Phe-Phe (Chelli et al., 2002; Joseph et al., 2021). A final data point of interest is the extremely low solubility of Tyr, over an order of magnitude smaller than that of Phe (0.045 and 2.79 g/100 g of water, respectively; see Figure 1D; Nozaki and Tanford, 1971). This low solubility has led to the suggestion that Tyr hydrogen bonds are stronger in the protein interior than those formed in water (Pace et al., 2001).

Bibliographic values for different properties of phenylalanine and tyrosine.

(A) Solvation free energy (ΔGsolv) (Chang et al., 2007). (B) Probability distributions of min-maxed normalized hydropathy values λ from bibliographic hydrophobicity scales (Tesei et al., 2021). (C) Self-interaction energy (εii) from the Miyazawa-Jernigan contact matrix (Miyazawa and Jernigan, 1996). (D) Solubility in water at 25°C (Nozaki and Tanford, 1971).

In summary, experimental results on condensates containing Phe/Tyr variants do not seem to align with either solvation free energies, most hydrophobicity scales, or statistical contact potentials, but are consistent with calculations with atomistic force fields in solution and solubilities in water. One possible explanation for these conflicting findings is that, due to their level of hydration, molecular condensates may differ significantly from the tightly-packed cores of folded protein structures (Lin et al., 2017; Das et al., 2020). Here, we address this paradox using a combination of classical molecular dynamics (MD) simulations and quantum chemical calculations. First, we estimate transfer free energies of peptides including these aromatic residues into model peptide condensates, and find that they are more favourable for Tyr than for Phe, an effect that is reversed when we perform the same transformation in apolar media. DFT calculations confirm that the interaction energies in Tyr-Tyr pairs are stronger than those between Phe residues. However, the transfer free energy contribution dominates at sufficiently low dielectric constants, making Phe-Phe pairs more favourable. These findings recapitulate the right rank order of interaction strengths of aromatic stickers in biomolecular condensates, but also their crossover in low dielectric media like the hydrophobic cores of folded proteins.