pH 效应可以主导 1H,15N-HSQC NMR 光谱中的化学位移扰动，用于研究小分子/α-突触核蛋白相互作用,ACS Chemical Neuroscience

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pH 效应可以主导 1H,15N-HSQC NMR 光谱中的化学位移扰动，用于研究小分子/α-突触核蛋白相互作用
ACS Chemical Neuroscience ( IF 4.1 ) Pub Date : 2023-02-07 , DOI: 10.1021/acschemneuro.2c00782
Anil K Pandey ₁ , Caroline R Buchholz ₂ , Noah Nathan Kochen ₁ , William C K Pomerantz _{2,

3} , Anthony R Braun ₁ , Jonathan N Sachs ₁

Affiliation

¹小时, ¹⁵N-异核单量子相干 (HSQC) NMR 是一种强大的技术，已用于表征小分子与本质无序的单体 α-突触核蛋白 (aSyn) 的相互作用。我们报告了溶液 pH 值如何影响 aSyn HSQC NMR 谱的解释，并证明小分子制剂（例如与酸性盐络合）可以降低样品 pH 值并混淆药物结合和伴随的蛋白质结构变化的解释。通过严格的 pH 控制，我们确认了几种先前鉴定的化合物（EGCG、黄芩苷和多巴胺 (DOPA)）以及一系列有效的 aSyn 病理学小分子抑制剂（地美环素、Ro90-7501 和 (±)-Bay） K 8644）能够直接与 aSyn 目标交战。之前，DOPA-aSyn 相互作用已被证明会在低 DOPA 浓度下引起剧烈的化学位移扰动 (CSP)，该化学位移扰动集中于 aSyn 的 H50，然后在增加的 DOPA 浓度下扩展到 aSyn 的酸性 C 末端残基。有趣的是，这个 CSP 曲线反映了我们的 pH 滴定，其中 pH 值的小幅降低会影响 H50 CSP，而 pH 值的大变化会导致 C 端 CSP 的稳定。相比之下，在严格控制 pH 5.0 的情况下，DOPA 会在可电离和不可电离残留物处观察到显着的 CSP。这些结果表明，先前对 DOPA-aSyn 相互作用的解释与 pH 诱导的 CSP 混为一谈，强调需要严格的 pH 控制，以尽量减少 HSQC NMR 实验中配体相互作用的潜在假阳性解释。此外，

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

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.

更新日期：2023-02-07

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