Piai Alessandro, Fu Qingshan, Dev Jyoti, Chou James J
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, 02115, USA.
Chemistry. 2017 Jan 26;23(6):1361-1367. doi: 10.1002/chem.201604206. Epub 2016 Dec 22.
Structural characterization of transmembrane proteins in isotropic bicelles has become an increasingly popular application of solution NMR spectroscopy, as the fast-tumbling bicelles are membrane-like, yet can often yield spectral quality comparable to those of detergent micelles. While larger bicelles are closer to the true lipid bilayer, it remains unclear how large the bicelles need to be to allow accurate assessment of the protein transmembrane partition in the lipid bilayer. Here, we address the above question from the perspective of the protein residing in the bicelles, through systematic measurement of the protein chemical shift and transmembrane partition at different lipid/detergent ratios (q), ranging from 0.3 to 0.7, using the transmembrane domain of the human Fas receptor as model system. We found that the lipid environment of the bicelles, as reflected by the protein chemical shift, begins to be perturbed when q is reduced to below 0.6. We also implemented a solvent paramagnetic relaxation enhancement (PRE) approach for bicelles to show that the protein transmembrane partition in bicelles with q=0.5 and 0.7 are very similar, but at q=0.3 the solvent PRE profile is significantly different. Our data indicate that q values between 0.5 and 0.6 are a good compromise between high resolution NMR and closeness to the membrane environment, and allow accurate characterization of the protein position in the lipid bilayer.
各向同性双分子层中跨膜蛋白的结构表征已成为溶液核磁共振波谱中越来越受欢迎的应用，因为快速翻滚的双分子层具有膜样性质，但通常能产生与去污剂胶束相当的光谱质量。虽然较大的双分子层更接近真实的脂质双层，但尚不清楚双分子层需要多大才能准确评估脂质双层中蛋白质的跨膜分配情况。在这里，我们以驻留在双分子层中的蛋白质为视角，通过使用人Fas受体的跨膜结构域作为模型系统，系统测量在0.3至0.7的不同脂质/去污剂比例（q）下蛋白质的化学位移和跨膜分配，来解决上述问题。我们发现，当q降至0.6以下时，由蛋白质化学位移反映的双分子层脂质环境开始受到扰动。我们还对双分子层实施了溶剂顺磁弛豫增强（PRE）方法，以表明q = 0.5和0.7的双分子层中蛋白质的跨膜分配非常相似，但在q = 0.3时，溶剂PRE谱有显著差异。我们的数据表明，q值在0.5至0.6之间是高分辨率核磁共振与接近膜环境之间的良好折衷，并且能够准确表征脂质双层中蛋白质的位置。
