Sušac Lukas, Horst Reto, Wüthrich Kurt
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (USA).
Chembiochem. 2014 May 5;15(7):995-1000. doi: 10.1002/cbic.201300729. Epub 2014 Apr 1.
X-ray crystallography and solution NMR of detergent-reconstituted OmpA (outer membrane protein A from E. coli) had shown that this protein forms an eight-stranded transmembrane β-barrel, but only limited information was obtained for the extracellular loops. In NMR studies of OmpA in two different detergent micelles, "NMR-invisible" amino acid residues in-between the extracellular loops and the β-barrel prevented complete structural characterization. Here, we show that this NMR-invisible ring around the β-barrel of OmpA is also present in lipid bilayer nanodiscs and in mixed micelles with a third detergent, thus suggesting that the implicated rate processes have a functional role rather than representing an artifact of the protein reconstitution. In addition to sequence-specific NMR assignments for OmpA in the nanodiscs, the present results are based on a protocol of micro-coil TROSY- and CRINEPT-type NMR diffusion measurements for studying the hydrodynamic properties and the foldedness of [(2)H,(15)N]-labeled membrane proteins in nanodiscs. This protocol can be applied under conditions closely similar to those used for NMR structure determinations or crystallization trials.
对去污剂重构的OmpA（大肠杆菌外膜蛋白A）进行的X射线晶体学和溶液核磁共振研究表明，该蛋白形成了一个由八条链组成的跨膜β桶，但关于细胞外环的信息有限。在对处于两种不同去污剂胶束中的OmpA进行的核磁共振研究中，细胞外环与β桶之间的“核磁共振不可见”氨基酸残基阻碍了完整的结构表征。在此，我们表明，OmpA的β桶周围这个核磁共振不可见的环也存在于脂质双层纳米盘中以及与第三种去污剂形成的混合胶束中，这表明所涉及的速率过程具有功能作用，而非代表蛋白质重构的假象。除了对纳米盘中OmpA进行序列特异性核磁共振归属外，本研究结果基于一种微线圈TROSY和CRINEPT型核磁共振扩散测量方案，用于研究[(2)H,(15)N]标记的纳米盘中膜蛋白的流体动力学性质和折叠情况。该方案可在与用于核磁共振结构测定或结晶试验的条件非常相似的条件下应用。
