Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratories, P.O. Box 9502, 2300 RA, Leiden, The Netherlands.
J Am Chem Soc. 2010 Aug 25;132(33):11487-95. doi: 10.1021/ja100867c.
Recent experimental studies have confirmed a long-held view that protein complex formation proceeds via a short-lived encounter state. The population of this transient intermediate, stabilized mainly by long-range electrostatic interactions, varies among different complexes. Here we show that the occupancy of the encounter state can be modulated across a broad range by single point mutations of interfacial residues. Using a combination of Monte Carlo simulations and paramagnetic relaxation enhancement NMR spectroscopy, we illustrate that it is possible to both enhance and diminish the binding specificity in an electron transfer complex of yeast cytochrome c (Cc) and cytochrome c peroxidase. The Cc T12A mutation decreases the population of the encounter to 10% as compared with 30% in the wild-type complex. More dramatically, the Cc R13A substitution reverses the relative occupancies of the stereospecific and the encounter forms, with the latter now being the dominant species with the population of 80%. This finding indicates that the encounter state can make a large contribution to the stability of a protein complex. Also, it appears that by adjusting the amount of the encounter through a judicious choice of point mutations, we can remodel the energy landscape of a protein complex and tune its binding specificity.
最近的实验研究证实了一个长期以来的观点，即蛋白质复合物的形成是通过短暂的相遇状态进行的。这种瞬态中间体的种群主要由长程静电相互作用稳定，在不同的复合物之间有所不同。在这里，我们表明通过界面残基的单点突变可以在很大范围内调节相遇状态的占有率。我们使用蒙特卡罗模拟和顺磁弛豫增强 NMR 光谱学的组合，说明了在酵母细胞色素 c (Cc) 和细胞色素 c 过氧化物酶的电子转移复合物中，既可以增强也可以减弱结合特异性。与野生型复合物中 30%的占有率相比，Cc T12A 突变将相遇状态的种群降低到 10%。更显著的是，Cc R13A 取代改变了立体特异性和相遇形式的相对占有率，后者现在是主要的形式，其种群为 80%。这一发现表明，相遇状态可以对蛋白质复合物的稳定性做出很大的贡献。此外，通过明智地选择点突变来调整相遇的数量，似乎可以重塑蛋白质复合物的能量景观并调整其结合特异性。
