Quantitative Measurement of Multiprotein Nanoparticle Interactions Using NMR Spectroscopy.
Quantitative Measurement of Multiprotein Nanoparticle Interactions Using NMR Spectroscopy.
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利用核磁共振光谱法对多蛋白纳米颗粒相互作用进行定量测量。
DOI：
10.1021/acs.analchem.1c01911
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发表时间：
2021-09-07
影响因子：
7.4
通讯作者：
Fitzkee NC
中科院分区：
文献类型：
--
作者：
Xu JX;Alom MS;Fitzkee NC
有效的基于强度的参考是定量核磁共振研究的基石，因为分子浓度是在其信号中编码的。从理论上讲，核磁共振非常适合于测量竞争性蛋白质在纳米颗粒(NP)表面的吸附，但目前的参比系统并不适合多维实验。本文提出了一种使用15N色氨酸(Trp)作为1H-15N 2D核磁共振实验的外部参照物的简单而新颖的参比系统。通过测定单个蛋白质与金纳米颗粒的结合能力，验证了该参比体系的有效性。然后，色氨酸参考被应用于蛋白质混合物，来自每种蛋白质的信号被准确地量化。所有结果与前人的研究结果一致，但具有更高的精度，表明Trp参照物可以准确地校准残留峰强度，并减少系统误差。最后，利用所提出的Trp参照物对不同蛋白质的竞争吸附进行了实时动态监测。作为一个具有挑战性的测试案例，我们成功地将我们的方法应用于只有一个残基不同的蛋白质变体的混合物。我们的结果表明，一种蛋白质的结合会影响另一种蛋白质的结合，导致NP日冕成分的改变。因此，这项工作突出了在原位研究蛋白质混合物中蛋白质-NP相互作用的重要性，并且这里开发的参比系统能够使用各种1H-15N 2D核磁共振技术来量化多个蛋白质的结合动力学和热力学。
An effective intensity-based reference is a cornerstone for quantitative nuclear magnetic resonance (NMR) studies, as the molecular concentration is encoded in its signal. In theory, NMR is well suited for the measurement of competitive protein adsorption onto nanoparticle (NP) surfaces, but current referencing systems are not optimized for multidimensional experiments. Presented herein is a simple and novel referencing system using 15N tryptophan (Trp) as an external reference for 1H–15N 2D NMR experiments. The referencing system is validated by the determination of the binding capacity of a single protein onto gold NPs. Then, the Trp reference is applied to protein mixtures, and signals from each protein are accurately quantified. All results are consistent with previous studies, but with substantially higher precision, indicating that the Trp reference can accurately calibrate the residue peak intensities and reduce systematic errors. Finally, the proposed Trp reference is used to kinetically monitor in situ and in real time the competitive adsorption of different proteins. As a challenging test case, we successfully apply our approach to a mixture of protein variants differing by only a single residue. Our results show that the binding of one protein will affect the binding of the other, leading to an altered NP corona composition. This work therefore highlights the importance of studying protein–NP interactions in protein mixtures in situ, and the referencing system developed here enables the quantification of binding kinetics and thermodynamics of multiple proteins using various 1H–15N 2D NMR techniques.
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影响因子：
6.1
作者：
Anthis NJ;Clore GM
通讯作者：
Clore GM
影响因子：
15
作者：
Ceccon A;Tugarinov V;Bax A;Clore GM
通讯作者：
Clore GM
影响因子：
13.3
作者：
Chanana, Munish;Correa-Duarte, Miguel A.;Liz-Marzan, Luis M.
通讯作者：
Liz-Marzan, Luis M.
DOI：
10.1002/anie.202008175
发表时间：
2020-12-21
期刊：
Angewandte Chemie (International ed. in English)
影响因子：
--
作者：
Pinals RL;Yang D;Rosenberg DJ;Chaudhary T;Crothers AR;Iavarone AT;Hammel M;Landry MP
通讯作者：
Landry MP
影响因子：
2.2
作者：
PALMER, AG;CAVANAGH, J;RANCE, M
通讯作者：
RANCE, M