调整固态 DNP 方法以研究 α-synuclein LLPS。

IF 3.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
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引用次数: 0

摘要

动态核极化(DNP)是一种利用电子自旋的量子感应能力来提高核磁共振(NMR)信号灵敏度的技术,尤其适用于不敏感的样品。玻璃化剂在 DNP 过程中起着至关重要的作用,它可以促进极化从未配对的电子自旋转移到核自旋,同时对生物大分子进行低温保护。在过去的二十年中,由甘油-d8/D2O/H2O 组成的 DNP 胶液已被广泛用于这一目的。聚乙二醇(PEG)也被用作低温保护剂,在模拟细胞条件的实验装置中,尤其是在体外制备液-液相分离(LLPS)冷凝物时,它经常被用作拥挤剂。在本研究中,我们研究了 PEG 在 DNP 果汁中替代甘油的功效,这对增强信号至关重要。改良后的 DNP 基质具有很高的 DNP 增强效果,因此可以通过固态 DNP 方法直接研究 LLPS 冷凝物,而无需添加任何外部成分。使用 PEG 的一个间接优势是 PEG 信号出现在 ∼72.5 ppm 处,与蛋白质光谱的脂肪族区域相对分离。使用 PEG 与水的混合物作为玻璃化剂,ASYMPOL-POK 作为最先进的极化剂,在不进行任何氘化的情况下,13C-甘氨酸的 DNP 得到了很大的交叉效应增强。DNP 增强效果和累积率与使用 DNP 果汁得到的结果相似,这表明在 LLPS 研究中,PEG 既是诱导拥挤的理想候选物质,也是玻璃化剂。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Tailoring solid-state DNP methods to the study of α-synuclein LLPS

Tailoring solid-state DNP methods to the study of α-synuclein LLPS

Dynamic Nuclear Polarization (DNP) is a technique that leverages the quantum sensing capability of electron spins to enhance the sensitivity of nuclear magnetic resonance (NMR) signals, especially for insensitive samples. Glassing agents play a crucial role in the DNP process by facilitating the transfer of polarization from the unpaired electron spins to the nuclear spins along with cryoprotection of biomolecules. DNPjuice comprising of glycerol-d8/D2O/H2O has been extensively used for this purpose over the past two decades. Polyethylene glycol (PEG), also used as a cryoprotectant, is often used as a crowding agent in experimental setups to mimic cellular conditions, particularly the invitro preparation of liquid-liquid phase separated (LLPS) condensates. In this study, we investigate the efficacy of PEG as an alternative to glycerol in the DNP juice, critical for signal enhancement. The modified DNP matrix leads to high DNP enhancement which enables direct study of LLPS condensates by solid-state DNP methods without adding any external constituents. An indirect advantage of employing PEG is that the PEG signals appear at 72.5 ppm and are relatively well-separated from the aliphatic region of the protein spectra. Large cross-effect DNP enhancement is attained for 13C-glycine by employing the PEG-water mixture as a glassing agent and ASYMPOL-POK as the state-of-art polarizing agent, without any deuteration. The DNP enhancement and the buildup rates are similar to results obtained with DNP juice, conforming to that PEG serves as a good candidate for both inducing crowding and glassing agent in the study of LLPS.

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来源期刊
Biophysical chemistry
Biophysical chemistry 生物-生化与分子生物学
CiteScore
6.10
自引率
10.50%
发文量
121
审稿时长
20 days
期刊介绍: Biophysical Chemistry publishes original work and reviews in the areas of chemistry and physics directly impacting biological phenomena. Quantitative analysis of the properties of biological macromolecules, biologically active molecules, macromolecular assemblies and cell components in terms of kinetics, thermodynamics, spatio-temporal organization, NMR and X-ray structural biology, as well as single-molecule detection represent a major focus of the journal. Theoretical and computational treatments of biomacromolecular systems, macromolecular interactions, regulatory control and systems biology are also of interest to the journal.
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