Methods to accelerate protein structure determination by solution NMR

蛋白质NMR化学位移对局部结构高度敏感。描述了一个强大的协议，利用这种关系从头蛋白质结构生成，作为输入实验参数的13钙，13 Cb，13 C，15 N，1Ha和1HN NMR化学位移。 这些位移通常在传统NMR结构测定过程的早期阶段，在收集和分析结构约束之前可用。基于化学位移的结构确定方案使用经验优化的程序从PDB中选择蛋白质片段，并结合标准ROSETTA蒙特卡罗组装和松弛方法。16个蛋白质，不同的大小从56到129个残基的评价产生了完整的原子模型，具有0.7-1.8均方根偏差的骨架原子相对于实验确定的X-射线或NMR结构。该策略也已成功地应用于9个蛋白质目标的分子量高达15.4 kDa，其常规的NMR结构测定是由东北结构基因组学联盟平行进行的盲法。 该方案为高通量NMR结构测定提供了一个新的方向。应用到同源二聚体蛋白质TolR，辅以小角度X射线散射数据，能够产生一个高分辨率的结构，这是几乎无法区分的结构，确定在一个耗时的方式使用传统的NMR策略。

Protein NMR chemical shifts are highly sensitive to local structure. A robust protocol is described that exploits this relation for de novo protein structure generation, using as input experimental parameters the 13Ca, 13Cb, 13C′, 15N, 1Ha and 1HN NMR chemical shifts. These shifts are generally available at the early stage of the traditional NMR structure determination process, prior to the collection and analysis of structural restraints. The chemical shift based structure determination protocol uses an empirically optimized procedure to select protein fragments from the PDB, in conjunction with the standard ROSETTA Monte Carlo assembly and relaxation methods. Evaluation of 16 proteins, varying in size from 56 to 129 residues yielded full atom models that have 0.7-1.8 root-mean-square deviations for the backbone atoms relative to the experimentally determined X-ray or NMR structures. The strategy also has been successfully applied in a blind manner to nine protein targets with molecular weights up to 15.4 kDa, whose conventional NMR structure determination was conducted in parallel by the Northeast Structural Genomics Consortium. This protocol potentially provides a new direction for high-throughput NMR structure determination. Application to the homodimeric protein TolR, supplemented by small angle Xray scattering data, was able to generate a high-resolution structure that was virtually indistinguishable from the structure determined in a time-consuming manner using the conventional NMR strategy.