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

Anil K. Pandey, Caroline R. Buchholz, Noah Nathan Kochen, William C.K. Pomerantz, Anthony R. Braun, Jonathan N. Sachs

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)800-808
Number of pages9
JournalACS Chemical Neuroscience
Volume14
Issue number4
DOIs
StatePublished - Feb 15 2023

Bibliographical note

Funding Information:
All NMR experiments were performed at the Minnesota NMR facility (MNMR). The authors disclose receipt of the following financial support for the research, authorship, and/or publication of this article. This study was supported by US National Institutes of Health to J.N.S. (NINDS 1R01NS117968) and National Institutes of Health Chemistry–Biology Interface Training Grant to C.R.B. (NIGMS 5T32GM132029-02).

Publisher Copyright:
© 2023 American Chemical Society.

Keywords

  • H,N-HSQC NMR
  • IDPs
  • small-molecule binding
  • α-synuclein

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