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稳定氧化物旋转标签用于通过马莱胺处理对生物分子的结构和形状研究

Xi-Wei Wang¹, Xing Zhang¹, Chao-Yu Cui¹

¹State Key Laboratory of Elemento-organic Chemistry, Tianjin Key Laboratory of Biosensing and Molecular Recognition, College of Chemistry, Nankai University, Tianjin, 300071, China.

Chemistry (Weinheim an der Bergstrasse, Germany)

|June 24, 2023

概括

氧化物 (NO) 旋转基,对于生物分子研究至关重要,在细胞溶解物中使用maleimide稳定. 这种方法克服了由酶引起的不稳定性,使得在接近细胞内条件下能够进行精确的电子磁共振 (EPR) 测量.

科学领域:

生物物理学的生物物理.
生物化学生化学
频谱学是一种光谱学.

背景情况:

氧化物 (NO) 旋转基是研究生物分子结构,相互作用和动态的宝贵工具.
电子磁共振 (EPR) 应用受阻于复杂的生物环境中NO激素的不稳定性,如细胞溶解物.
之前的假设将NO的激素不稳定性归因于降解剂,如谷氨 (GSH),但这项研究将NADPH/NADH依赖酶确定为主要原因.

研究的目的:

确定导致细胞溶解物中NO旋转基不稳定的因素.
开发一种稳定细胞溶解物中的NO基的方法,以实现长期EPR测量.
为了证明这种稳定方法在接近细胞内条件下研究生物分子的实用性.

主要方法:

研究了细胞溶解物中NO基的不稳定性,测试了各种潜在的药物,包括降解试剂和maleimide.
确定了依赖NADPH/NADH的酶是NO基的不稳定性的原因.
利用maleimide治疗通过消耗NADPH/NADH来稳定NO基,从而抑制破坏稳定的酶.
在经过处理的细胞溶解物中对NO标记蛋白进行了双电子-电子共振 (DEER) 测量.

主要成果:

证明依赖NADPH/NADH的酶,而不是GSH,会在细胞溶解物中破坏NO基的稳定.
通过向NADPH/NADH依赖酶,证明了马莱胺有效地稳定NO基.
证实,马利米德治疗保留了准确EPR测量所需的细胞内拥挤特性.
成功地将maleimide稳定策略应用于DEER对NO标记蛋白质的测量.

结论:

马莱胺治疗是稳定细胞溶解物中的NO旋转基的强有力的策略,克服了酶降解.
这种方法允许在非常接近细胞内环境的条件下对NO标记生物分子进行EPR研究.
这些发现扩大了NO自旋标签和EPR光谱的适用性,用于研究复杂生物矩阵中的生物分子系统.

关键词:

在DEER测量中测量了DEER.细胞溶解酸细胞溶解酸.蛋白质旋转标签蛋白质旋转标签蛋白质结构蛋白质结构对于NO基的稳定性

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