Transverse relaxation-optimized NMR spectroscopy with the outer membrane protein OmpX in dihexanoyl phosphatidylcholine micelles
Transverse relaxation-optimized NMR spectroscopy with the outer membrane protein OmpX in dihexanoyl phosphatidylcholine micelles
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DOI：
10.1073/pnas.051629298
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发表时间：
2001-02-27
影响因子：
11.1
通讯作者：
Wüthrich, K
中科院分区：
文献类型：
--
作者：
Fernández, C;Adeishvili, K;Wüthrich, K
用二己酰磷脂酰胆碱（DHPC）在分子量约为60 kDa的混合胶束中重组大肠杆菌（Escherichia coli）的H-2、C-13、N-15标记的148个残基的膜整合蛋白OmpX。在pH 6.8和30 ℃下，在这些混合胶束的2 mM水溶液中记录横向弛豫优化光谱（TROSY）型三重共振NMR实验和TROSY型核Overhauser增强光谱。因此，已经获得了多肽骨架的完整序列特异性NMR分配。C-13化学位移和核Overhauser效应数据，然后导致在溶液中的OmpX/DHPC的常规二级结构元素的识别和在收集的输入的构象约束的计算的全球折叠的蛋白质。相同类型的多肽骨架折叠中观察到目前确定的溶液结构和先前报道的晶体结构的OmpX在洗涤剂的存在下确定的正辛基四氧杂环丁烷。进一步的结构优化将不得不依赖于部分或完全质子化的氨基酸侧链的额外的共振分配，但目前的数据已经表明，弛豫优化的NMR技术为研究膜蛋白的结构和功能开辟了新的途径。
The H-2,C-13,N-15-labeled, 148-residue integral membrane protein OmpX from Escherichia coli was reconstituted with dihexanoyl phosphatidylcholine (DHPC) in mixed micelles of molecular mass of about 60 kDa. Transverse relaxation-optimized spectroscopy (TROSY)-type triple resonance NMR experiments and TROSY-type nuclear Overhauser enhancement spectra were recorded in 2 mM aqueous solutions of these mixed micelles at pH 6.8 and 30 degreesC. Complete sequence-specific NMR assignments for the polypeptide backbone thus have been obtained. The C-13 chemical shifts and the nuclear Overhauser effect data then resulted in the identification of the regular secondary structure elements of OmpX/DHPC in solution and in the collection of an input of conformational constraints for the computation of the global fold of the protein. The same type of polypeptide backbone fold is observed in the presently determined solution structure and the previously reported crystal structure of OmpX determined in the presence of the detergent n-octyltetraoxyethylene. Further structure refinement will have to rely on the additional resonance assignment of partially or fully protonated amino acid side chains, but the present data already demonstrate that relaxation-optimized NMR techniques open novel avenues for studies of structure and function of integral membrane proteins.