Abstract

Protein–protein interactions (PPIs) are crucial for the successful realization of many metabolic and signaling pathways. Of particular interest are PPIs of membrane proteins, which form stable and/or transient complexes for the signal transduction, ion transport, and electron transfer within electron transfer chains. The Surface Plasmon Resonance (SPR) allows analyzing the thermodynamic and kinetic parameters (k_on and k_off) and equilibrium constants (K_d) of PPI, as well as the assessment of the effects of low molecular weight compounds. The aim of the present study was the adaptation of the SPR protocols for PPIs involving mitochondrial cytochrome P450 CYP11A1, mitochondrial cytochrome b5 (CYB5B), and adrenodoxin (Adx). We found that the Adx–CYP11A1 and CYB5B–CYP11A interactions depend on the method of protein immobilization and on the microenvironment. For example, the k_off values for complexes Adx–CYP11A1 and CYB5B–CYP11A1 in the aqueous environment were similar (1.5 ± 0.2) × 10^–3 s^–1, while the values of k_on for these complexes differed from each other by almost one order of magnitude ((6.5 ± 0.5) × 10⁴ and (0.30 ± 0.03) × 10⁴ M^–1 s^–1, respectively). This is in good agreement with the known high affinity of CYP11A1 to its cognate redox partner Adx. In the lipid environment, the rate of complex dissociation was higher than that in the aqueous environment with k_off value equal to (9.1 ± 0.3) × 10^–3 s^–1. For the CYP11A1–CYB5B complex, the parameters of interaction in the lipid phase were not determined due to unspecific binding of the proteins used as analytes to the lipids immobilized on the L1 chip. We show for the first time that SPR method could be used for the detection and quantitative analysis of PPI involving mitochondrial cytochromes (CYP) with their redox partners in both aqueous and lipid environment. Experimental protocols developed and validated in our previous work and in this one can serve as valuable tools for studies of interactions of CYP proteins and could also be applied for other membrane proteins.

中文翻译：

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SPR生物传感器在水环境和双层脂质膜中蛋白质-蛋白质相互作用的分析中的应用，以P450scc（CYP11A1）为例

摘要

蛋白质间相互作用（PPI）对于成功实现许多代谢和信号通路至关重要。特别感兴趣的是膜蛋白的PPI，它们形成稳定的和/或瞬时的复合物，用于信号转移，离子传输和电子转移链内的电子转移。表面等离子体共振（SPR）可以分析PPI的热力学和动力学参数（k _on和k _off）和平衡常数（K _d），以及评估低分子量化合物的作用。本研究的目的是针对涉及线粒体细胞色素P450 CYP11A1，线粒体细胞色素b5的PPI调整SPR方案（CYB5B）和肾上腺素（Adx）。我们发现Adx–CYP11A1和CYB5B–CYP11A的相互作用取决于蛋白质固定方法和微环境。例如，在水环境中，复合物Adx–CYP11A1和CYB5B–CYP11A1的k _off值相似（1.5±0.2）×10 ^–3 s ^–1，而这些复合物的k _on值彼此之间几乎相差几乎一个数量级（（6.5±0.5）×10 ⁴和（0.30±0.03）×10 ⁴ M ^–1 s ^–1， 分别）。这与CYP11A1与其同源氧化还原伙伴Adx的已知高亲和力完全吻合。在脂质环境中，复合物的解离速率高于水性环境，其中k _off值等于（9.1±0.3）×10 ^–3 s ^–1。对于CYP11A1-CYB5B复合物，由于作为分析物的蛋白质与固定在L1芯片上的脂质的非特异性结合，因此未确定在脂质相中的相互作用参数。我们首次展示了SPR方法可用于在水性和脂质环境中检测和定量分析涉及线粒体细胞色素（CYP）及其氧化还原伙伴的PPI。在我们之前的工作中开发并验证的实验规程，在此实验规程中可以用作研究CYP蛋白相互作用的有价值的工具，也可以用于其他膜蛋白。