Pi-Pi contacts are an overlooked protein feature relevant to phase separation

Abstract

Protein phase separation is implicated in formation of membraneless organelles, signaling puncta and the nuclear pore. Multivalent interactions of modular binding domains and their target motifs can drive phase separation. However, forces promoting the more common phase separation of intrinsically disordered regions are less understood, with suggested roles for multivalent cation-pi, pi-pi, and charge interactions and the hydrophobic effect. Known phase-separating proteins are enriched in pi-orbital containing residues and thus we analyzed pi-interactions in folded proteins. We found that pi-pi interactions involving non-aromatic groups are widespread, underestimated by force-fields used in structure calculations and correlated with solvation and lack of regular secondary structure, properties associated with disordered regions. We present a phase separation predictive algorithm based on pi interaction frequency, highlighting proteins involved in biomaterials and RNA processing.

Arginine Side‐Chain Hydrogen Exchange: Quantifying Arginine Side‐Chain Interactions in Solution

“…Moreover, small changes in temperature and particularly in pH affect the rates substantially, thus generally allowing for a large range of arginine side‐chains to be characterised. It is envisaged that the new method serves as a particularly valuable tool to characterise active sites in enzymes,, protein‐protein or protein‐nucleic acid interactions, and phase separation, where arginine residues are expected to play a crucial role for biological function.…”

“…The arginine guanidinium group has a very high p K a of ∼14 and the delocalised positive charge is therefore present under all physiologically relevant pH values . Each arginine guanidinium group has a large delocalised π‐system for cation‐π and π−π interactions, as well as five guanidinium protons that are available for hydrogen‐bonding and salt‐bridging, but generally labile and able to exchange with the bulk solvent. The hydrogen exchange along with restricted rotation about the C ζ −N ϵ bond are unfortunately often a hinderance to obtaining conventional 1 H‐ 15 N NMR correlation spectra at neutral or high‐pH ,.…”

RNAs, Phase Separation, and Membrane‐Less Organelles: Are Post‐Transcriptional Modifications Modulating Organelle Dynamics?

“…Proteins prone to undergoing LLPS are either multi‐domain proteins or harbor intrinsically disordered regions (IDRs), often also referred to as prion‐like domains (PLDs) . Individual IDRs can self‐associate and form oligomeric structures through multiple weak and adhesive interactions such as cation‐pi, pi‐stacking, or dipolar interactions between stretches of particular “disorder‐promoting” amino acids (i.e., Q, S, N, Y, G), all resulting in phase separation of interacting proteins from surrounding solution . The ensuing multi‐valency governs inter‐ and intramolecular interactions resulting in the assembly of oligo‐ and polymeric structures in which valency (the number of interaction modules) and affinity (strength of interactions of individual modules) are the key parameters controlling LLPS.…”

Physical Chemistry of Cellular Liquid‐Phase Separation

“…In addition, π quadrupoles can similarly associate with other dipoles or quadrupoles, and may play a role in carbohydrate‐π interactions by attraction to polarized C−H bonds . π–π stacking interactions have been suggested as a predictor of phase separation capacity …”

“…π systems are common in proteins, both in aromatic side chains and in the sp 2 ‐hybridized atoms of residues like glutamine, asparagine, and arginine . Peptide bonds also contain sp 2 ‐hybridized atoms, which are relevant for small residues that expose those bonds for interaction . π interactions are important both in protein structure and interactions among intrinsically disordered proteins.…”