McIntosh L P, Wand A J, Lowry D F, Redfield A G, Dahlquist F W
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Biochemistry. 1990 Jul 10;29(27):6341-62. doi: 10.1021/bi00479a003.
The proton and nitrogen (15NH-H alpha-H beta) resonances of bacteriophage T4 lysozyme were assigned by 15N-aided 1H NMR. The assignments were directed from the backbone amide 1H-15N nuclei, with the heteronuclear single-multiple-quantum coherence (HSMQC) spectrum of uniformly 15N enriched protein serving as the master template for this work. The main-chain amide 1H-15N resonances and H alpha resonances were resolved and classified into 18 amino acid types by using HMQC and 15N-edited COSY measurements, respectively, of T4 lysozymes selectively enriched with one or more of alpha-15N-labeled Ala, Arg, Asn, Asp, Gly, Gln, Glu, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val. The heteronuclear spectra were complemented by proton DQF-COSY and TOCSY spectra of unlabeled protein in H2O and D2O buffers, from which the H beta resonances of many residues were identified. The NOE cross peaks to almost every amide proton were resolved in 15N-edited NOESY spectra of the selectively 15N enriched protein samples. Residue specific assignments were determined by using NOE connectivities between protons in the 15NH-H alpha-H beta spin systems of known amino acid type. Additional assignments of the aromatic proton resonances were obtained from 1H NMR spectra of unlabeled and selectively deuterated protein samples. The secondary structure of T4 lysozyme indicated from a qualitative analysis of the NOESY data is consistent with the crystallographic model of the protein.
通过¹⁵N辅助的¹H NMR对噬菌体T4溶菌酶的质子和氮（¹⁵NH-Hα-Hβ）共振进行了归属。这些归属是从主链酰胺¹H-¹⁵N核开始的，均匀¹⁵N富集蛋白的异核单多重量子相干（HSMQC）谱作为这项工作的主模板。分别使用¹⁵N编辑的COSY测量和HMQC，对选择性富集一种或多种α-¹⁵N标记的丙氨酸、精氨酸、天冬酰胺、天冬氨酸、甘氨酸、谷氨酰胺、谷氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸或缬氨酸的T4溶菌酶，解析并将主链酰胺¹H-¹⁵N共振和Hα共振分类为18种氨基酸类型。通过未标记蛋白在H₂O和D₂O缓冲液中的质子DQF-COSY和TOCSY谱对异核谱进行补充，从中鉴定出许多残基的Hβ共振。在选择性¹⁵N富集蛋白样品的¹⁵N编辑NOESY谱中解析了几乎每个酰胺质子的NOE交叉峰。通过已知氨基酸类型的¹⁵NH-Hα-Hβ自旋系统中质子之间的NOE连接性确定残基特异性归属。从未标记和选择性氘代蛋白样品的¹H NMR谱中获得了芳香族质子共振的额外归属。从NOESY数据的定性分析得出的T4溶菌酶二级结构与该蛋白的晶体学模型一致。
