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Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

Published on: November 22, 2014

在选择性甲基标记蛋白中进行自动分配.

Yingqi Xu¹, Minhao Liu, Peter J Simpson

¹Division of Molecular Biosciences, Cross Faculty NMR Centre, Imperial College London, South Kensington, London, SW7 2AZ, UK.

Journal of the American Chemical Society

|June 19, 2009

概括

本研究引入了一种自动化方法,用于使用NMR光谱学在大型蛋白质中分配甲基. 这种技术显著加快了该过程,将99%的甲基组分配给没有突变的甲基组.

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科学领域:

生物化学生物化学
结构生物学结构生物学
核磁共振 (NMR) 光谱学核磁共振 (NMR) 光谱学

背景情况:

核磁共振 (NMR) 光谱是确定蛋白质结构和动态的强大工具.
横向放松优化光谱学 (TROSY) 和特定的甲基标记方案使得对较大的蛋白质 (高达1MDa) 的NMR研究成为可能.
目前用于在大型蛋白质中的甲基团共振分配的方法依赖于耗时的突变发生或碎片分析,由于化学转移干扰,这可能使解释复杂化.

研究的目的:

开发和验证一种新型的自动化程序,用于在非常大的蛋白质中快速分配甲基团共振.
克服现有的耗时且可能造成混的基于突变发生的策略的局限性.
通过NMR实现大型蛋白质复合物的高效结构和动态研究.

主要方法:

开发一种用于甲基组分配的自动计算程序.
该方法应用于标有异黄素,素和白素 (ILV) 的300kDa蛋白酶体复合体.
使用大蛋白系统上记录的高质量的NMR光谱.

主要成果:

自动化程序迅速分配了300kDa蛋白酶体中的大多数甲基.
在没有人工干预的情况下,99%的观察到的甲基组被正确分配.
该方法成功地绕过了对突变发生或碎片分析的需求.

结论:

开发的自动化程序通过NMR显著加快了对非常大的蛋白质的甲基组分配.
这种方法为传统方法提供了更有效和可靠的替代方案,促进了对大型生物分子系统的研究.
该工具是公开可用的 (http://nmr.bc.ic.ac.uk/map-xs/),用于更广泛的科学应用.