Tugarinov Vitali, Kay Lewis E
Protein Engineering Network Centre of Excellence and the Department of Medical Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
J Am Chem Soc. 2003 May 14;125(19):5701-6. doi: 10.1021/ja021452+.
A sensitive 3D NMR pulse scheme, (H)C(CA)NH-COSY, is presented for the assignment of (13)C(delta)(1) Ile chemical shifts in large perdeuterated, methyl-protonated proteins. The nonlinearity of branched amino acids, such as Ile, significantly degrades the quality of TOCSY schemes which transfer magnetization from methyl carbons to the backbone (13)C(alpha) positions, and in applications to high molecular weight proteins (correlation times on the order of 40-50 ns), this compromises the sensitivity of spectra used for methyl assignment. The experiment presented utilizes COSY-based transfer steps and refocuses undesirable (13)C-(13)C scalar couplings that degrade the efficiency of TOCSY transfers. The (H)C(CA)NH-COSY scheme is tested on an (15)N,(13)C,(2)H-[Leu, Val, Ile (delta 1 only)]-methyl-protonated maltose binding protein (MBP)/beta-cyclodextrin complex at 5 degrees C (molecular tumbling time 46 +/- 2 ns), facilitating the assignment of (13)C(delta 1) chemical shifts for 18 of the 19 Ile residues for which backbone assignments were previously obtained. Both sensitivity and resolution of the resulting spectra are shown to be significantly better than those for a similar TOCSY-based approach.
本文提出了一种灵敏的3D NMR脉冲序列（H）C（CA）NH-COSY，用于在大型全氘化、甲基质子化蛋白质中确定（13）C（δ）1异亮氨酸的化学位移。支链氨基酸（如异亮氨酸）的非线性会显著降低将磁化从甲基碳转移到主链（13）C（α）位置的TOCSY序列的质量，在应用于高分子量蛋白质（相关时间约为40-50 ns）时，这会损害用于甲基归属的光谱的灵敏度。本文提出的实验利用基于COSY的转移步骤，并重新聚焦那些会降低TOCSY转移效率的不良（13）C-（13）C标量耦合。（H）C（CA）NH-COSY序列在5℃下对（15）N，（13）C，（2）H-[亮氨酸、缬氨酸、异亮氨酸（仅δ1）]-甲基质子化麦芽糖结合蛋白（MBP）/β-环糊精复合物进行了测试（分子翻滚时间为46±2 ns），有助于确定19个异亮氨酸残基中18个的（13）C（δ1）化学位移，此前已获得这些残基的主链归属。结果光谱的灵敏度和分辨率均显示明显优于类似的基于TOCSY的方法。
