在小乳酸中1型铜中心的轴联体突变的意想不到的影响:基于结构的分析和工程来增加减少潜力和活性
Jing-Xiang Wang1, Avery C Vilbert2, Lucas H Williams3
1Department of Chemistry, The University of Texas at Austin 105 East 24th Street Austin TX 78712 USA yi.lu@utexas.edu.
多铜氧化酶中的1型铜 (T1Cu) 中心的蛋白质工程是复杂的. 小乳糖酶 (SLAC) 的新突变成功调整了T1Cu的降解潜力和活性,用于增强电子转移应用.
科学领域:
- 生物化学和生物物理学
- 蛋白质工程是指蛋白质工程.
- 生物有机化学 生物有机化学
背景情况:
- 1型铜 (T1Cu) 中心对于生物电子转移 (ET) 至关重要,可调节的还原潜力 (E) 对于高效的氧化还原反应至关重要.
- 在单核蛋白中调整T1Cu中心已经确立,但由于复杂的结构因素,在多铜氧化酶中具有挑战性.
- 在小乳糖酶 (SLAC) 中的一种甲因向白氨酸突变 (M298L) 意外地降低了它的E,这归因于轴性水分子和降低的疏水性.
研究的目的:
- 调查M298L-SLAC中降低E的结构基础,并确定增强T1Cu中调的策略.
- 通过基于结构的蛋白质设计来改进SLAC的降解潜力和催化活性.
- 为生物技术应用提供T1Cu中心调在多铜氧化酶中的复杂性提供见解.
主要方法:
- 用X射线结晶学分析突变对T1Cu中心的结构影响.
- 分子动力学模拟以了解轴环位置和水分子的影响.
- 位点定向突变发生 (F195L和I200F) 来设计SLAC并评估E和催化效率 (kcat/KM) 的变化.
主要成果:
- 在SLAC中的M298L突变导致E因轴性水分子的存在而降低,与单核蛋白不同.
- 结构分析显示T1Cu轴环位置的差异导致了不同的突变结果.
- 组合突变 (M298L,F195L,I200F) 部分消除了轴水,使E增加了122mV,并增加了kcat/KM的7倍.
结论:
- 多铜氧化酶中T1Cu中心的蛋白质工程是复杂的,需要考虑轴环动力学和水合.
- 结构引导突变可以成功调整T1Cu E,并增强SLAC中的催化活性.
- 这些发现为设计用于太阳能,燃料电池和生物修复的T1Cu蛋白提供了宝贵的见解.
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