Backbone conformational constraints in a microcrystalline U-15N-Labeled protein by 3D dipolar-shift solid-state NMR spectroscopy
Backbone conformational constraints in a microcrystalline U-15N-Labeled protein by 3D dipolar-shift solid-state NMR spectroscopy
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DOI：
10.1021/ja058292x
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发表时间：
2006-03-15
影响因子：
15
通讯作者：
Rienstra, CM
中科院分区：
文献类型：
--
作者：
Franks, WT;Wylie, BJ;Rienstra, CM
近年来，通过魔角旋转核磁共振波谱法对均匀标记的蛋白质进行结构研究已经迅速成熟。几种蛋白质的位点特异性化学位移已被分配和结构确定从2D或3D数据集包含核间距离信息。在这里，我们展示了一个互补的技术，用于约束蛋白质骨架的几何形状，使用一个网站解决的3D偶极位移脉冲序列的应用。偶极线的形状反映了1H −15N[i]到1H −15N[i+1]偶极矢量的相对取向，限制了扭转角θ [i]和θ [i]。此外，从相同的3D数据集中，提取几个1H − 15 N [i]至H − 15 N [i+2]线形以约束扭转角θ [i]、θ [i]、θ [i+1]和θ [i+1]。我们报告了在蛋白G（GB1）的56个残基的β1免疫球蛋白结合结构域中的大多数位点的结果，使用600 MHz 1H频率的3D实验。SSNMR结果和一个新的1.14 μ m晶体结构之间的极好的一致性说明了这种技术用于固体蛋白质的高分辨率结构细化的一般潜力。
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 of1H−15N[i] to1H−15N[i+1] dipole vectors, constraining the torsion angles ϕ[i] and ψ[i]. In addition, from the same 3D data set, several1H−15N[i] toH−15N[i+2] line shapes are extracted to constrain the torsion angles ϕ[i], ψ[i], ϕ[i+1], and ψ[i+1]. We report results for the majority of sites in the 56-residue β1 immunoglobulin binding domain of protein G (GB1), using 3D experiments at 600 MHz1H frequency. Excellent agreement between the SSNMR results and a new 1.14 Å crystal structure illustrate the general potential of this technique for high-resolution structural refinement of solid proteins.