Magnetic Resonance Center, CERM, University of Florence, Via L. Sacconi, 6-50019 Sesto Fiorentino, Italy.
J Am Chem Soc. 2010 Jan 27;132(3):1032-40. doi: 10.1021/ja906426p.
The use of pseudocontact shifts arising from paramagnetic metal ions in a microcrystalline protein sample is proposed as a strategy to obtain unambiguous signal assignments in solid-state NMR spectra enabling distance extraction for protein structure calculation. With this strategy, 777 unambiguous (281 sequential, 217 medium-range, and 279 long-range) distance restraints could be obtained from PDSD, DARR, CHHC, and the recently introduced PAR and PAIN-CP solid-state experiments for the cobalt(II)-substituted catalytic domain of matrix metalloproteinase 12 (159 amino acids, 17.6 kDa). The obtained structure is a high resolution one, with backbone rmsd of 1.0 +/- 0.2 A, and is in good agreement with the X-ray structure (rmsd to X-ray 1.3 A). The proposed strategy, which may be generalized for nonmetalloproteins with the use of paramagnetic tags, represents a significant step ahead in protein structure determination using solid-state NMR.
提出了一种利用顺磁金属离子在微结晶蛋白样品中产生的赝接触位移的策略，以获得固态 NMR 谱中明确的信号分配，从而能够提取距离用于蛋白质结构计算。使用该策略，从钴（II）取代的基质金属蛋白酶 12（159 个氨基酸，17.6 kDa）的催化结构域的 PDSD、DARR、CHHC 和最近引入的 PAR 和 PAIN-CP 固态实验中，可以获得 777 个明确的（281 个序列，217 个中程和 279 个远程）距离约束。得到的结构是一个高分辨率的结构，主链 rmsd 为 1.0 +/- 0.2 A，与 X 射线结构（与 X 射线的 rmsd 为 1.3 A）非常吻合。该策略可以通过使用顺磁标记物推广到非金属蛋白酶，代表了使用固态 NMR 进行蛋白质结构测定的重要一步。
