通过三维和四维魔角旋转固态核磁共振光谱法测定肽和蛋白质中的甲基13C-15N偶极耦合。

Determination of methyl 13C-15N dipolar couplings in peptides and proteins by three-dimensional and four-dimensional magic-angle spinning solid-state NMR spectroscopy.

作者信息

Helmus Jonathan J, Nadaud Philippe S, Höfer Nicole, Jaroniec Christopher P

机构信息

Department of Chemistry, The Ohio State University, Columbus, Ohio 43210, USA.

出版信息

J Chem Phys. 2008 Feb 7;128(5):052314. doi: 10.1063/1.2817638.

DOI:10.1063/1.2817638

PMID:18266431

Abstract

We describe three- and four-dimensional semiconstant-time transferred echo double resonance (SCT-TEDOR) magic-angle spinning solid-state nuclear magnetic resonance (NMR) experiments for the simultaneous measurement of multiple long-range (15)N-(13)C(methyl) dipolar couplings in uniformly (13)C, (15)N-enriched peptides and proteins with high resolution and sensitivity. The methods take advantage of (13)C spin topologies characteristic of the side-chain methyl groups in amino acids alanine, isoleucine, leucine, methionine, threonine, and valine to encode up to three distinct frequencies ((15)N-(13)C(methyl) dipolar coupling, (15)N chemical shift, and (13)C(methyl) chemical shift) within a single SCT evolution period of initial duration approximately 1(1)J(CC) (where (1)J(CC) approximately 35 Hz, is the one-bond (13)C(methyl)-(13)C J-coupling) while concurrently suppressing the modulation of NMR coherences due to (13)C-(13)C and (15)N-(13)C J-couplings and transverse relaxation. The SCT-TEDOR schemes offer several important advantages over previous methods of this type. First, significant (approximately twofold to threefold) gains in experimental sensitivity can be realized for weak (15)N-(13)C(methyl) dipolar couplings (corresponding to structurally interesting, approximately 3.5 A or longer, distances) and typical (13)C(methyl) transverse relaxation rates. Second, the entire SCT evolution period can be used for (13)C(methyl) and/or (15)N frequency encoding, leading to increased spectral resolution with minimal additional coherence decay. Third, the experiments are inherently "methyl selective," which results in simplified NMR spectra and obviates the use of frequency-selective pulses or other spectral filtering techniques. Finally, the (15)N-(13)C cross-peak buildup trajectories are purely dipolar in nature (i.e., not influenced by J-couplings or relaxation), which enables the straightforward extraction of (15)N-(13)C(methyl) distances using an analytical model. The SCT-TEDOR experiments are demonstrated on a uniformly (13)C, (15)N-labeled peptide, N-acetyl-valine, and a 56 amino acid protein, B1 immunoglobulin-binding domain of protein G (GB1), where the measured (15)N-(13)C(methyl) dipolar couplings provide site-specific information about side-chain dihedral angles and the packing of protein molecules in the crystal lattice.

摘要

我们描述了三维和四维半恒定时间转移回波双共振（SCT-TEDOR）魔角旋转固态核磁共振（NMR）实验，用于在均匀（13）C、（15）N富集的肽和蛋白质中同时高分辨率和高灵敏度地测量多个长程（15）N-（13）C（甲基）偶极耦合。这些方法利用了丙氨酸、异亮氨酸、亮氨酸、蛋氨酸、苏氨酸和缬氨酸中侧链甲基的（13）C自旋拓扑结构，在初始持续时间约为1（1）J（CC）（其中（1）J（CC）约为35Hz，是一键（13）C（甲基）-（13）C J耦合）的单个SCT演化期内编码多达三个不同频率（（15）N-（13）C（甲基）偶极耦合、（15）N化学位移和（13）C（甲基）化学位移），同时抑制由于（13）C-（13）C和（15）N-（13）C J耦合以及横向弛豫引起的NMR相干调制。与以前的此类方法相比，SCT-TEDOR方案具有几个重要优点。首先，对于弱（15）N-（13）C（甲基）偶极耦合（对应于结构上有趣的、约3.5埃或更长的距离）和典型的（13）C（甲基）横向弛豫率，可以实现实验灵敏度的显著（约两倍到三倍）提高。其次，整个SCT演化期可用于（13）C（甲基）和/或（15）N频率编码，从而在最小的额外相干衰减情况下提高光谱分辨率。第三，这些实验本质上是“甲基选择性的”，这导致NMR光谱简化，无需使用频率选择性脉冲或其他光谱滤波技术。最后，（15）N-（13）C交叉峰积累轨迹本质上纯粹是偶极的（即不受J耦合或弛豫影响），这使得能够使用分析模型直接提取（15）N-（13）C（甲基）距离。在均匀（13）C、（15）N标记的肽N-乙酰缬氨酸和56个氨基酸的蛋白质G的B1免疫球蛋白结合结构域（GB1）上展示了SCT-TEDOR实验，其中测量的（15）N-（13）C（甲基）偶极耦合提供了关于侧链二面角和蛋白质分子在晶格中堆积的位点特异性信息。