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通过自上而下紫外光光解离质谱法对二叶酸减少酶复合物的结构性表征

Michael B Cammarata¹, Ross Thyer¹, Jake Rosenberg¹

¹†Department of Chemistry and ‡Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, Texas 78712, United States.

Journal of the American Chemical Society

|July 1, 2015

概括

193纳米的UVPD质谱法追踪了二叶酸减少酶 (DHFR) 的结构变化. 这项技术绘制了NADPH和甲状腺酸盐 (MTX) 的联结位点,揭示了其催化循环期间的酶灵活性.

科学领域:

生物化学生物化学
结构生物学结构生物学
质谱测量质量谱测量

背景情况:

在将二叶酸降解为四叶酸的过程中,二叶酸减少酶 (DHFR) 经历了显著的结构变化.
了解这些动态变化对于理解酶功能和设计向抑制剂至关重要.

研究的目的:

使用193 nm UVPD,在辅因子NADPH和抑制剂甲基酸盐 (MTX) 的存在下描述DHFR的构造格局.
在DHFR中映射NADPH和MTX的结合点,并将碎片化模式与形状重排联系起来.

主要方法:

193纳米紫外光解离 (UVPD) 质谱法用于分析二进制 (DHFR·NADPH,DHFR·MTX) 和三进制 (DHFR·NADPH·MTX) 复合体.
获得了广泛的序列覆盖,产生了碎片离子,这些离子表明了连接体相互作用和构造状态.

主要成果:

UVPD提供了高序列覆盖率 (>80%) 并揭示了联体结合的片段,将NADPH映射到腺结合域和MTX映射到循环域.
碎片化模式显示了脊柱裂解倾向的显著变化,反映了像子域旋转和结时的循环运动等构造变化.
特定区域表现出增强的裂变 (增加灵活性) 或抑制的裂变 (连接体屏蔽/分子内相互作用).

结论:

193纳米UVPD是一种多功能和灵敏的技术,用于监测催化循环期间的酶构造动态.
该研究成功地将UVPD碎片化与已知的DHFR结构和功能方面相关联,验证了该方法的实用性.
结果提供了关于三元复合体和酶灵活性过渡状态的见解.

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