Conformational Changes and Drivers of Monoclonal Antibody Liquid-Liquid Phase Separation.
Conformational Changes and Drivers of Monoclonal Antibody Liquid-Liquid Phase Separation.
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单克隆抗体液-液相分离的构象变化和驱动因素。
DOI：
10.1016/j.xphs.2022.10.017
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发表时间：
2023
影响因子：
3.8
通讯作者：
Middaugh,CRussell
中科院分区：
文献类型：
--
作者：
Larson,NicholasR;Wei,Yangjie;Cruz,ThayanaAraújo;Esfandiary,Reza;Kalonia,CavanK;Forrest,MLaird;Middaugh,CRussell
液-液相分离是生物学中的一种现象，蛋白质可以分离成具有不同性质的密相和更稀的相。三种经过液-液相分离的抗体在富蛋白相和贫蛋白相中进行了表征。与贫蛋白质相相比，富蛋白质相表现出更多的蓝移色氨酸发射和红移酰胺 I 吸光度。使用拉曼光谱观察到涉及二硫键周围构象异构化的巨大变化。各相之间的酰胺 I 和蛋白质荧光差异持续到高于临界温度的温度，但在发生聚集的温度时停止。此外，所有三种抗体的富含蛋白质相内的水分子的结构组织都发生了很大的变化。据推测，由于蛋白质在两个相中具有相同的化学势，因此富含蛋白质的相中的蛋白质粘度较高，导致该相中扩散依赖性蛋白质聚集减慢。对于所有三种抗体，我们进行了加速稳定性研究，发现富含蛋白质的相以与缺乏蛋白质的相相同的速度或更慢的速度聚集。
Liquid-liquid phase separation is a phenomenon within biology whereby proteins can separate into dense and more dilute phases with distinct properties. Three antibodies that undergo liquid-liquid phase separation were characterized in the protein-rich and protein-poor phases. In comparison to the protein-poor phase, the protein-rich phase demonstrates more blue-shift tryptophan emissions and red-shifted amide I absorbances. Large changes involving conformational isomerization around disulfide bonds were observed using Raman spectroscopy. Amide I and protein fluorescence differences between the phases persisted to temperatures above the critical temperature but ceased at the temperature at which aggregation occurred. In addition, large changes occurred in the structural organization of water molecules within the protein-rich phase for all three antibodies. It is hypothesized that as the proteins have the same chemical potential in both phases, the protein viscosity is higher in the protein-rich phase resulting in slowed diffusion dependent protein aggregation in this phase. For all three antibodies we performed accelerated stability studies and found that the protein-rich phase aggregated at the same rate or slower than the protein-poor phase.