Lescop Ewen, Brutscher Bernhard
Laboratoire de Chimie et Biologie Structurales, Institut de Chimie des Substances Naturelles, CNRS UPR 2301, 1, Avenue de la Terrasse, 91190, Gif-sur-Yvette, France.
J Biomol NMR. 2009 May;44(1):43-57. doi: 10.1007/s10858-009-9314-2. Epub 2009 Apr 15.
Sequential resonance assignment represents an essential step towards the investigation of protein structure, dynamics, and interaction surfaces. Although the experimental sensitivity has significantly increased in recent years, with the availability of high field magnets and cryogenically cooled probes, resonance assignment, even of small globular proteins, still generally requires several days of data collection and analysis using standard protocols. Here we introduce the BATCH strategy for fast and highly automated backbone resonance assignment of (13)C, (15)N-labelled proteins. BATCH makes use of the fast data acquisition and analysis tools BEST, ASCOM, COBRA, and HADAMAC, recently developed in our laboratory. An improved Hadamard encoding scheme, presented here, further increases the performance of the HADAMAC experiment. A new software platform, interfaced to the NMRView software package, has been developed that enables highly automated NMR data processing and analysis, sequential resonance assignment, and (13)C chemical shift extraction. We demonstrate for four small globular proteins that sequential resonance assignment can be routinely obtained within a few hours, or less, in a highly automated and robust way.
序列共振归属是研究蛋白质结构、动力学和相互作用表面的关键步骤。尽管近年来实验灵敏度显著提高，随着高场磁体和低温冷却探头的出现，即使是小分子球状蛋白质的共振归属，使用标准方案通常仍需要几天的数据收集和分析。在此，我们介绍用于（13）C、（15）N标记蛋白质快速且高度自动化的主链共振归属的BATCH策略。BATCH利用了我们实验室最近开发的快速数据采集和分析工具BEST、ASCOM、COBRA和HADAMAC。本文提出的改进的哈达玛编码方案进一步提高了HADAMAC实验的性能。已开发出一个与NMRView软件包接口的新软件平台，该平台能够进行高度自动化的NMR数据处理和分析、序列共振归属以及（13）C化学位移提取。我们证明，对于四种小分子球状蛋白质，可以通过高度自动化且稳健的方式在几小时或更短时间内常规获得序列共振归属。
