Franks W Trent, Wylie Benjamin J, Stellfox Sara A, Rienstra Chad M
Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
J Am Chem Soc. 2006 Mar 15;128(10):3154-5. doi: 10.1021/ja058292x.
Structural studies of uniformly labeled proteins by magic-angle spinning NMR spectroscopy have rapidly matured in recent years. Site-specific chemical shifts of several proteins have been assigned and structures determined from 2D or 3D data sets containing internuclear distance information. Here we demonstrate the application of a complementary technique for constraining protein backbone geometry using a site-resolved 3D dipolar-shift pulse sequence. The dipolar line shapes report on the relative orientations of 1H-15N[i] to 1H-15N[i+1] dipole vectors, constraining the torsion angles phi[i] and psi[i]. In addition, from the same 3D data set, several 1H-15N[i] to1H-15N[i+2] line shapes are extracted to constrain the torsion angles phi[i], psi[i], phi[i+1], and psi[i+1]. We report results for the majority of sites in the 56-residue beta1 immunoglobulin binding domain of protein G (GB1), using 3D experiments at 600 MHz 1H frequency. Excellent agreement between the SSNMR results and a new 1.14 A crystal structure illustrate the general potential of this technique for high-resolution structural refinement of solid proteins.
近年来，通过魔角旋转核磁共振光谱对均匀标记蛋白质进行的结构研究迅速成熟。已经确定了几种蛋白质的位点特异性化学位移，并根据包含核间距离信息的二维或三维数据集确定了结构。在这里，我们展示了一种互补技术的应用，该技术使用位点分辨的三维偶极位移脉冲序列来约束蛋白质主链几何结构。偶极线形反映了1H-15N[i]与1H-15N[i+1]偶极矢量的相对取向，从而约束扭转角phi[i]和psi[i]。此外，从同一个三维数据集中，提取了几个从1H-15N[i]到1H-15N[i+2]的线形，以约束扭转角phi[i]、psi[i]、phi[i+1]和psi[i+1]。我们报告了使用600 MHz 1H频率的三维实验对蛋白质G（GB1）的56个残基β1免疫球蛋白结合结构域中大多数位点的研究结果。固体核磁共振结果与新的1.14 Å晶体结构之间的出色一致性说明了该技术在固体蛋白质高分辨率结构优化方面的一般潜力。
