黄蛋白作为细胞内ESR光谱中的原生和遗传编码的自旋探针
Timothée Chauviré1,2, Siddarth Chandrasekaran1, Robert Dunleavy1
1Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA.
Nature communications
|July 2, 2025
概括
电子自旋共振 (ESR) 光谱检测揭示了细菌的原生膜中的空中吸附受体 (Aer) 的结构. 这种方法还引入了一种新的光-氧和电压 (LOV) 领域,用于研究细胞中的蛋白质结构.
科学领域:
- 生物物理学的生物物理.
- 结构生物学 结构生物学
- 微生物学 微生物学
背景情况:
- 弗拉辅因子作为有效的电子旋转共振 (ESR) 探针,因为它们能够通过细胞减光剂和光线形成半子状态.
- 细菌的跨膜空中吸收受体 (Aer) 在细胞导航和信号转导中发挥着至关重要的作用.
研究的目的:
- 通过ESR光谱学研究Aer受体在其原生Escherichia coli膜环境中的结构性质和寡合体状态.
- 开发一种基因编码的光-氧和电压 (LOV) 域,作为一种多功能ESR探针,用于研究细胞环境中的蛋白质结构.
主要方法:
- 为ESR测量优化光谱条件 (电子放松时间) 和细胞生长 (同位素标记).
- 使用连续波ESR,Q波段电子核双共振 (ENDOR) 和Q波段四脉冲双电子-电子共振 (4P-DEER) 光谱.
- 开发并将基因编码的LOV域纳入目标蛋白.
主要成果:
- 在ESR研究中,证实了在辅因子口袋中固定化的刚性Aer flavin,并确定了占主导地位的阳离子半农基态.
- 4P-DEER测量确定了Aer同位素黄之间的4.1纳米距离,并揭示了以前未被观察到的分离,表明化学受体阵列在现场.
- 证明了工程 LOV 域作为用于细胞内部结构分析的 ESR 探针的实用性.
结论:
- ESR光谱学为细菌细胞内的Aer受体的原生结构和相互作用提供了宝贵的见解.
- 开发的LOV域提供了一种新且广泛适用的工具,用于在体内阐明蛋白质结构动态和寡合状态.
- 这种研究框架有助于研究复杂的蛋白质组合和结构,这些组合和结构很难在体外复制.
相关概念视频
01:19Protein Dynamics in Living Cells
2.3K
Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
2.3K
03:31Labeling DNA Probes
8.4K
DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
8.4K