Meghan N. Ricciardi

The utility of intrinsically disordered regions (IDRs) in protein function has become an area of broad interest in recent years given their known roles in signaling, biomolecular condensates, and protein activity regulation. The central challenge, however, is that disorder is difficult to characterize, and thus, there is a need to develop approaches to monitor how IDRs influence proteins. Here, we employ a strategy to restrict the freedom of the intrinsically disordered N-terminal tail (ID-tail) of human thymidylate synthase (hTS), which, together with tail truncation, enables testing aspects of how the ID-tail modulates enzyme function. The highly flexible hTS tail is known to impact allosteric substrate cooperativity and conformational switching dynamics, although how the tail brings about these effects has not been determined. We attempted to restrict native tail dynamics by chemically linking the dimer tails and tested linked forms with activity, binding, and nuclear magnetic resonance heteronuclear single quantum coherence (NMR HSQC) spectra. Truncating four N-terminal residues eliminated cooperativity in dUMP binding to apo-hTS, but because a similar result was obtained from the linked full-length protein, complete tail freedom appeared to be important for intersubunit communication. NMR analysis was aided by referencing the inactive M190K mutant, which roughly defined regions of active-inactive conformational switching that were disrupted by tail linkage. Collectively, these results point toward a role of the ID-tail to influence the ordered core of the protein. Moreover, while the natural ID-tail is extremely flexible, it likely possesses specific physiochemical movement dynamics and positioning attributes that facilitate its impact on allosteric cooperativity and conformational dynamics.

Reader proteins that bind histone trimethyllysine (Kme3) have become active therapeutic targets in recent years. Binding of Kme3 in the conserved aromatic cages of these readers via cation-π interactions is a key contributor to these protein-protein interactions. We explored whether the replacement of one methyl group of Kme3 with an electron-withdrawing group (EWG) could improve binding by increasing the electrostatic component of the cation-π interaction. We examined these Kme3 analogs in binding studies with histone reader proteins, nuclear magnetic resonance (NMR) studies in a β-hairpin peptide model system, and computational studies to gain mechanistic insight into their binding. Kme3 analogs with both alkyl and benzyl substitution with EWGs were examined. We found that EWGs on the ligand differentially affected binding to a series of Kme3 readers but did not improve binding. This is likely due to sterics of the different analogs that preclude optimal cation-π interactions. The unique selectivity patterns of these ligands nonetheless offer promise toward selective inhibitor development within this protein class. NMR studies of the alkyl-substituted analogs in the β-hairpin model system show that EWGs strengthen the cation-π interaction enthalpically but with an entropic penalty. NMR studies demonstrate that the benzyl group makes direct contacts with Trp in the β-hairpin model, providing aromatic interactions in addition to cation-π interactions. Computational energy decomposition analysis confirms the more favorable electrostatic interactions compared to Kme3, but other factors counter the enhanced electrostatic component. This mechanistic investigation demonstrates how EWG-containing Kme3 analogs contribute to cation-π interactions and provide unique selectivity patterns relative to Kme3.

Impeded by the complexity of proteinaceous structure and the very weak nature of the noncovalent interactions involved, the detailed mechanisms by which anions induce salting-in Hofmeister effects in proteins and peptides remain unclear. Here, using β-hairpin peptides as models, we examine two approaches to qualify (map) anion binding: ¹H NMR chemical shifts and hydronium-catalyzed hydrogen-deuterium exchange (HDX) rate changes. We demonstrate that each salt investigated─despite an affinity too weak to quantify accurately, caused denaturation to an extent that is both peptide and anion-specific, with more charge-diffuse anions inducing a greater degree of unfolding. Our studies reveal that the HDX mapping provides more detail than chemical shift data. Thus, HDX mapping reveals two slightly different mechanisms of denaturation, depending on the nature of the anion. Namely, assisted by a N-terminal Arg residue, charge-dense Cl^- is chelated by the terminal N-H groups of the hairpin and induces a small degree of denaturation, whereas charge-diffuse anions intercalate deeply into the cation-π-hydrophobic core of the peptide and induce more substantial unfolding. These findings provide a glimpse of the different mechanisms by which anions can induce the salting-in Hofmeister effect in peptides and proteins and suggest HDX as a useful tool to map weak anion binding.

Per-residue analysis of the thermodynamics of unfolding of β-hairpins provides insight into the non-covalent interactions between residues, and the individual contributions of residues and secondary structure type to the Δ H , Δ S , and Δ C _p of unfolding.