University at Albany, Department of Chemistry, Albany, NY, 12222, USA.
Georgia Tech, School of Mechanical Engineering, Atlanta, GA, 30332, USA.
Commun Biol. 2022 May 12;5(1):451. doi: 10.1038/s42003-022-03412-x.
High-resolution structural studies of proteins and protein complexes in a native eukaryotic environment present a challenge to structural biology. In-cell NMR can characterize atomic resolution structures but requires high concentrations of labeled proteins in intact cells. Most exogenous delivery techniques are limited to specific cell types or are too destructive to preserve cellular physiology. The feasibility of microfluidics transfection or volume exchange for convective transfer, VECT, as a means to deliver labeled target proteins to HeLa cells for in-cell NMR experiments is demonstrated. VECT delivery does not require optimization or impede cell viability; cells are immediately available for long-term eukaryotic in-cell NMR experiments. In-cell NMR-based drug screening using VECT was demonstrated by collecting spectra of the sensor molecule DARPP32, in response to exogenous administration of Forskolin.
在天然真核环境中对蛋白质和蛋白质复合物进行高分辨率结构研究对结构生物学提出了挑战。在细胞内 NMR 可以对原子分辨率结构进行表征，但需要在完整细胞中高浓度标记的蛋白质。大多数外源递呈技术仅限于特定的细胞类型，或者对维持细胞生理学过于破坏。本文证明了微流控转染或体积交换作为一种对流转移（VECT）的方法，用于将标记的靶蛋白递送到 HeLa 细胞中进行在细胞内 NMR 实验的可行性。VECT 递呈不需要优化，也不会妨碍细胞活力；细胞可立即用于长期的真核在细胞内 NMR 实验。通过收集传感器分子 DARPP32 的光谱，证明了 VECT 用于在细胞内 NMR 的药物筛选。
