pH Effects Can Dominate Chemical Shift Perturbations in ¹H,¹⁵N-HSQC NMR Spectroscopy for Studies of Small Molecule/α-Synuclein Interactions

Abstract

1H,15N-Heteronuclear Single Quantum Coherence (HSQC) NMR is a powerful technique that has been employed to characterize small-molecule interactions with intrinsically disordered monomeric α-Synuclein (aSyn). We report how solution pH can impact the interpretation of aSyn HSQC NMR spectra and demonstrate that small-molecule formulations (e.g., complexation with acidic salts) can lower sample pH and confound interpretation of drug binding and concomitant protein structural changes. Through stringent pH control, we confirm that several previously identified compounds (EGCG, Baicalin, and Dopamine (DOPA)) as well as a series of potent small-molecule inhibitors of aSyn pathology (Demeclocycline, Ro90-7501, and (±)-Bay K 8644) are capable of direct target engagement of aSyn. Previously, DOPA–aSyn interactions have been shown to elicit a dramatic chemical shift perturbation (CSP) localized to aSyn’s H50 at low DOPA concentrations then expanding to aSyn’s acidic C-terminal residues at increasing DOPA levels. Interestingly, this CSP profile mirrors our pH titration, where a small reduction in pH affects H50 CSP, and large pH changes induce robust C-terminal CSP. In contrast, under tightly controlled pH 5.0, DOPA induces significant CSPs observed at both ionizable and nonionizable residues. These results suggest that previous interpretations of DOPA–aSyn interactions were conflated with pH-induced CSP, highlighting the need for stringent pH control to minimize potential false-positive interpretations of ligand interactions in HSQC NMR experiments. Furthermore, DOPA’s preferential interaction with aSyn under acidic pH represents a novel understanding of DOPA–aSyn interactions that may provide insight into the potential gain of toxic function of aSyn misfolding in α-synucleinopathies.

Picosecond Dynamics of a Small Molecule in Its Bound State with an Intrinsically Disordered Protein

“…Furthermore, it has recently been reported that heteronuclear single-quantum coherence spectra (HSQCs) of disordered proteins are highly prone to falsepositive characterization of ligand interactions due to artifacts arising from mismatched pH. 36 In contrast, we report here that ligand-detected 19 F transverse relaxation measurements are sensitive to small-molecule/IDP binding.…”

“…The largest protein-detected chemical shift perturbations we observe (more than 1 SD across all residues, each less than 12 ppb), which are consistent across two concentrations of 5-fluoro­indole, occur at residues A308, W312, A313, T334, S414, and S448. Furthermore, it has recently been reported that heteronuclear single-quantum coherence spectra (HSQCs) of disordered proteins are highly prone to false-positive characterization of ligand interactions due to artifacts arising from mismatched pH . In contrast, we report here that ligand-detected 19 F transverse relaxation measurements are sensitive to small-molecule/IDP binding.…”

The combination of inorganic phosphate and pyrophosphate 31P‐NMR for the electrodeless pH determination in the 5–12 range

“…The insertion of the glass electrode in the NMR tube at each addition would progressively alter the total volume making the titration largely inaccurate. Unless a very effective buffer can be used, chemical shift variations due to pH changes would overlap with those due to other sources, possibly leading to results misinterpretation 8 . NMR pH‐sensing methods must be used to monitor pH evolution in‐situ.…”

Fragment-Based NMR Screening of the BPTF PHD Finger Methyl Lysine Reader Leads to the First Small-Molecule Inhibitors

“…Additionally, H34 was most significantly perturbed; however, H34 perturbation could potentially be due to a pH effect. 31 Future crystallization studies will aid in the development of more elaborated fragments with higher affinity and will be useful for assessing how these scaffolds interact with the BPTF PHD.…”

“…The last fragment, F4 , which we suspected to be a promiscuous binder or potential denaturant through Zn chelation, did not show evidence of denaturing the protein, and aromatic cage residues (Y17 and Y23) were perturbed one standard deviation from the average CSP (Figure S13). Additionally, H34 was most significantly perturbed; however, H34 perturbation could potentially be due to a pH effect . Future crystallization studies will aid in the development of more elaborated fragments with higher affinity and will be useful for assessing how these scaffolds interact with the BPTF PHD.…”