Institute of Chemistry, Leiden University, The Netherlands.
Computational Biochemistry, University of Bayreuth, Germany.
FEBS J. 2022 Jan;289(2):535-548. doi: 10.1111/febs.16159. Epub 2021 Sep 13.
Optimal charge distribution is considered to be important for efficient formation of protein complexes. Electrostatic interactions guide encounter complex formation that precedes the formation of an active protein complex. However, disturbing the optimized distribution by introduction of extra charged patches on cytochrome c peroxidase does not lead to a reduction in productive encounters with its partner cytochrome c. To test whether a complex with a high population of encounter complex is more easily affected by suboptimal charge distribution, the interactions of cytochrome c mutant R13A with wild-type cytochrome c peroxidase and a variant with an additional negative patch were studied. The complex of the peroxidase and cytochrome c R13A was reported to have an encounter state population of 80%, compared to 30% for the wild-type cytochrome c. NMR analysis confirms the dynamic nature of the interaction and demonstrates that the mutant cytochrome c samples the introduced negative patch. Kinetic experiments show that productive complex formation is fivefold to sevenfold slower at moderate and high ionic strength values for cytochrome c R13A but the association rate is not affected by the additional negative patch on cytochrome c peroxidase, showing that the total charge on the protein surface can compensate for less optimal charge distribution. At low ionic strength (44 mm), the association with the mutant cytochrome c reaches the same high rates as found for wild-type cytochrome c, approaching the diffusion limit.
最佳电荷分布被认为对于高效形成蛋白质复合物很重要。静电相互作用指导了在形成活性蛋白质复合物之前的复合物形成的遭遇。然而，在细胞色素 c 过氧化物酶上引入额外的带电荷斑块并不会导致与伴侣细胞色素 c 的有效遭遇减少，从而破坏了优化的分布。为了测试具有高遭遇复合物的复合物是否更容易受到次优电荷分布的影响，研究了细胞色素 c 突变体 R13A 与野生型细胞色素 c 过氧化物酶以及带有额外负电荷斑块的变体的相互作用。据报道，过氧化物酶与细胞色素 c R13A 的复合物的遭遇状态群体为 80%，而野生型细胞色素 c 的遭遇状态群体为 30%。NMR 分析证实了相互作用的动态性质，并表明突变细胞色素 c 采样了引入的负电荷斑块。动力学实验表明，在中等和高离子强度值下，细胞色素 c R13A 的有效复合物形成速度慢五倍至七倍，但细胞色素 c 过氧化物酶上的额外负电荷斑块不会影响缔合速率，表明蛋白质表面的总电荷可以补偿次优电荷分布。在低离子强度（44 mM）下，与突变细胞色素 c 的缔合速度达到与野生型细胞色素 c 相同的高速度，接近扩散限制。
