Methods to accelerate protein structure determination by solution NMR

蛋白质核磁共振化学位移对局部结构高度敏感。描述了一个健壮的方案，它利用这种关系来从头生成蛋白质结构，使用13Ca，13Cb，13C和15N，1HA和1HN核磁共振化学位移作为输入参数。这些位移通常在传统核磁共振结构确定过程的早期阶段可用，在收集和分析结构限制之前。基于化学位移的结构确定方法使用经验性优化的程序从PDB中选择蛋白质片段，并结合标准的Rosetta蒙特卡罗组装和松弛方法。对16种蛋白质的评估，从56个残基到129个残基不等，得到了完整的原子模型，相对于实验确定的X射线或核磁共振结构，主链原子的均方根偏差为0.7-1.8。该策略还成功地盲法应用于9个分子量高达15.4 kDa的蛋白质靶标，其常规核磁共振结构测定是由东北结构基因组联合会并行进行的。该方法为高通量核磁共振结构的确定提供了新的方向。将其应用于同二聚体蛋白质TolR，并辅之以小角X射线散射数据，能够产生与传统核磁共振策略以耗时方式确定的结构几乎无法区分的高分辨率结构。

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.