Research Center for Next-Generation Protein Sciences, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan; Graduate School of Pharmaceutical Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Department of Molecular Biophysics, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Biophys Chem. 2024 Dec;315:107328. doi: 10.1016/j.bpc.2024.107328. Epub 2024 Sep 18.
The observation of side-chain peaks of aromatic amino acids is the prerequisite for a high-resolution three-dimensional structure determination of proteins by NMR. However, it becomes difficult with increasing molecular size due to an increased transverse relaxation and the control of the relaxation pathway is needed to achieve the observation. We demonstrated that even for the large molecular size of 82 kDa Malate synthase G (MSG), the aromatic C-H (CH) peaks of Tryptophan (Trp) and Phenylalanine (Phe) residues can be observed with high quality using a systematic stable isotope labeling scheme, Stereo-Array Isotope Labeling (SAIL) method. However, the sequence specific assignments of these peaks relied on the use of amino acid substitutions, employing an inefficient method that required many isotopes labeled samples. In this study, we developed novel SAIL amino acids that allow for the observation of the aromatic ring δ,ζ and the aliphatic β position peak of Phe residues. The application of TROSY-based experiment to the isolated CH moieties resulted in the successful observation of discernible and resolved CH peaks in Phe residues in MSG. In MSG, the sequence-specific assignments of the backbone and C positions have already been confirmed. Therefore, using this labeling method, the δ and β position peaks of Phe residues can be clearly assigned in a sequence-specific and stereospecific manner through experiments based on intra-residue NOE. Furthermore, the NOESY experiment also allows for the acquisition of information pertaining to the conformation of Phe residues, such as the χ1 dihedral angle, providing valuable insights for the determination of accurate protein structures and in dynamic analysis. This new SAIL amino acids open an avenue to achieve a variety of NMR analysis of large molecular proteins, including a high-resolution structure determination and dynamics and interaction analysis.
芳香族氨基酸侧链峰的观测是通过 NMR 对蛋白质进行高分辨率三维结构测定的前提。然而，由于横向弛豫的增加，随着分子尺寸的增加，这变得更加困难，需要控制弛豫途径来实现观测。我们证明，即使对于分子量为 82 kDa 的苹果酸合酶 G（MSG）这样的大分子，也可以使用系统的稳定同位素标记方案——立体阵列同位素标记（SAIL）方法，高质量地观测到色氨酸（Trp）和苯丙氨酸（Phe）残基的芳香族 C-H（CH）峰。然而，这些峰的序列特异性分配依赖于使用氨基酸取代，采用效率低下的方法，需要许多同位素标记的样品。在这项研究中，我们开发了新型 SAIL 氨基酸，允许观测 Phe 残基的芳香环 δ、ζ 和脂肪族 β 位峰。基于 TROSY 的实验应用于分离的 CH 部分，导致 MSG 中 Phe 残基的 CH 峰可分辨且分辨率高。在 MSG 中，已经确认了骨架和 C 位置的序列特异性分配。因此，使用这种标记方法，可以通过基于残基内 NOE 的实验以序列特异性和立体特异性的方式清楚地分配 Phe 残基的 δ 和 β 位峰。此外，NOESY 实验还可以获取与 Phe 残基构象相关的信息，例如 χ1 二面角，为确定准确的蛋白质结构和动态分析提供有价值的见解。这种新的 SAIL 氨基酸为实现对大分子量蛋白质的各种 NMR 分析开辟了道路，包括高分辨率结构测定、动力学和相互作用分析。
