Leiden Institute of Physics, Huygens-Kamerlingh Onnes Laboratory, Leiden University, Niels Bohrweg 2, 2333 CA, Leiden (The, Netherlands.
Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratories, Einsteinweg 55, 2333 CC, Leiden (The, Netherlands.
Chemphyschem. 2020 May 18;21(10):1060-1069. doi: 10.1002/cphc.201901160. Epub 2020 Apr 17.
We present a novel approach to study transient protein-protein complexes with standard, 9 GHz, and high-field, 95 GHz, electron paramagnetic resonance (EPR) and paramagnetic NMR at ambient temperatures and in solution. We apply it to the complex of yeast mitochondrial iso-1-cytochrome c (Cc) with cytochrome c peroxidase (CcP) with the spin label [1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)-methanethiosulfonate] attached at position 81 of Cc (SL-Cc). A dissociation constant K of 20±4×10 M (EPR and NMR) and an equal amount of stereo-specific and encounter complex (NMR) are found. The EPR spectrum of the fully bound complex reveals that the encounter complex has a significant population (60 %) that shares important features, such as the Cc-interaction surface, with the stereo-specific complex.
我们提出了一种新的方法来研究瞬时蛋白-蛋白复合物，使用标准的 9GHz 和高场的 95GHz 电子顺磁共振（EPR）和顺磁 NMR，在环境温度和溶液中进行。我们将其应用于酵母线粒体同工型 1-细胞色素 c（Cc）与细胞色素 c 过氧化物酶（CcP）的复合物，其中自旋标记[1-氧代-2,2,5,5-四甲基-Δ3-吡咯啉-3-甲基）-甲硫磺酸酯]连接在 Cc 的位置 81（SL-Cc）。发现解离常数 K 为 20±4×10M（EPR 和 NMR）和等量的立体特异性和遭遇复合物（NMR）。完全结合的复合物的 EPR 谱表明，遭遇复合物具有显著的部分（60%），其与立体特异性复合物共享重要特征，如 Cc 相互作用表面。
