Zytkiewicz, E. | Search bioRxiv

bioRxiv2023

Quantifying Amide-Aromatic Interactions at Molecular and Atomic Levels: Experimentally-determined Enthalpic and Entropic Contributions to Interactions of Amide sp2O, N, C and sp3C Unified Atoms with Naphthalene sp2C Atoms in Water

In addition to amide hydrogen bonds and the hydrophobic effect, interactions involving {pi}-bonded sp2 atoms of amides, aromatics and other groups occur in protein self-assembly processes including folding, oligomerization and condensate formation. These interactions also occur in aqueous solutions of amide and aromatic compounds, where they can be quantified. Previous analysis of thermodynamic coefficients quantifying net-favorable interactions of amide compounds with other amides and aromatics revealed that interactions of amide sp2O with amide sp2N unified atoms (presumably C=O{middle dot}{middle dot}{middle dot}H-N hydrogen bonds) and amide/aromatic sp2C (lone pair-{pi}, n-{pi}*) are particularly favorable. Sp3C-sp3C (hydrophobic), sp3C-sp2C (hydrophobic, CH-{pi}), sp2C-sp2C (hydrophobic, {pi}-{pi}) and sp3C-sp2N interactions are favorable, sp2C-sp2N interactions are neutral, while sp2O-sp2O and sp2N-sp2N self-interactions and sp2O-sp3C interactions are unfavorable. Here, from determinations of favorable effects of fourteen amides on naphthalene solubility at 10, 25 and 45 {degrees}C, we dissect amide-aromatic interaction free energies into enthalpic and entropic contributions and find these vary systematically with amide composition. Analysis of these results yields enthalpic and entropic contributions to intrinsic strengths of interactions of amide sp2O, sp2N, sp2C and sp3C unified atoms with aromatic sp2C atoms. For each interaction, enthalpic and entropic contributions have the same sign and are much larger in magnitude than the interaction free energy itself. The amide sp2O-aromatic sp2C interaction is enthalpy-driven and entropically unfavorable, consistent with direct chemical interaction (e.g. lone pair-{pi}) while amide sp3C- and sp2C-aromatic sp2C interactions are entropy-driven and enthalpically unfavorable, consistent with hydrophobic effects. These findings are relevant for interactions involving {pi}-bonded sp2 atoms in protein processes. Table of Contents Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/548600v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1683bb4org.highwire.dtl.DTLVardef@27db75org.highwire.dtl.DTLVardef@b915ccorg.highwire.dtl.DTLVardef@1461b04_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

How Glutamate Promotes Liquid-liquid Phase Separation and DNA Binding Cooperativity of E. coli SSB Protein

E. coli single-stranded-DNA binding protein (EcSSB) displays nearest-neighbor (NN) and non-nearest-neighbor (NNN)) cooperativity in binding ssDNA during genome maintenance. NNN cooperativity requires the intrinsically-disordered linkers (IDL) of the C-terminal tails. Potassium glutamate (KGlu), the primary E. coli salt, promotes NNN-cooperativity, while KCl inhibits it. We find that KGlu promotes compaction of a single polymeric SSB-coated ssDNA beyond what occurs in KCl, indicating a link of compaction to NNN-cooperativity. EcSSB also undergoes liquid-liquid phase separation (LLPS), inhibited by ssDNA binding. We find that LLPS, like NNN-cooperativity, is promoted by increasing [KGlu] in the physiological range, while increasing [KCl] and/or deletion of the IDL eliminate LLPS, indicating similar interactions in both processes. From quantitative determinations of interactions of KGlu and KCl with protein model compounds, we deduce that the opposing effects of KGlu and KCl on SSB LLPS and cooperativity arise from their opposite interactions with amide groups. KGlu interacts unfavorably with the backbone (especially Gly) and side chain amide groups of the IDL and therefore promotes amide-amide interactions in LLPS and NNN-cooperativity. By contrast, KCl interacts favorably with these amide groups and therefore inhibits LLPS and NNN-cooperativity. These results highlight the importance of salt interactions in regulating the propensity of proteins to undergo LLPS.

biochemistry↗