Amplification of Nuclear Overhauser Effect Signals by Hyperpolarization for Screening of Ligand Binding to Immobilized Target Proteins
Amplification of Nuclear Overhauser Effect Signals by Hyperpolarization for Screening of Ligand Binding to Immobilized Target Proteins
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DOI：
10.1021/acs.analchem.0c01071
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发表时间：
2020-10-20
影响因子：
7.4
通讯作者：
Hilty, Christian
中科院分区：
文献类型：
--
作者：
Wang, Yunyi;Hilty, Christian
靶蛋白的固定化增强了核自旋极化转移的交叉弛豫速率，但降低了可达的靶浓度。溶解动态核极化（D-DNP）的配体自旋的超极化被证明可以增加观察配体核内Overhauser效应（NOE）的灵敏度。这种效应，也称为转移NOE（trNOE），可用于检测结合和获得结合相关的结构信息。超极化的trNOE信号的测量证明了配体4 '-羟基偶氮苯-2-羧酸与固定在聚苯乙烯珠上的抗生物素蛋白相互作用。在含有63.5 μ M配体和0.83 μ M可及蛋白质结合位点的样品中，由D-DNP提供的信号增强导致NOE积聚的单扫描检测，尽管该信号峰值仅为总配体信号的2%。这些累积曲线允许通过NOE符号的变化和交叉弛豫速率的定量测定来确认结合。D-DNP技术和蛋白质固定化的组合可能有助于在药物发现的配体筛选中鉴定配体内NOE。相同的方法可应用于配体与细胞表面蛋白相互作用的体内表征。
Immobilization of a target protein enhances the cross-relaxation rates for transfer of nuclear spin polarization but reduces the accessible target concentration. Hyperpolarization of ligand spins by dissolution dynamic nuclear polarization (D-DNP) is shown to increase sensitivity for observing the intraligand nuclear Overhauser effect (NOE). This effect, also known as the transferred NOE (trNOE), can be used for detection of binding and for obtaining binding-related structural information. The measurement of hyperpolarized trNOE signals is demonstrated for the ligand 4'-hydroxyazobenzene-2-carboxylic acid interacting with avidin protein immobilized on polystyrene beads. In a sample containing 63.5 mu M ligands and 0.83 mu M accessible protein binding sites, the signal enhancement provided by D-DNP leads to single-scan detection of the NOE buildup, despite that this signal peaks at only 2% of the total ligand signal. These buildup curves allow the confirmation of binding through a change in the sign of the NOE and the quantitative determination of cross-relaxation rates. The combination of the D-DNP technique and protein immobilization may facilitate the identification of intraligand NOEs in ligand screening for drug discovery. The same method may be applied to in vivo characterization of ligand interactions with cell surface proteins.