Pacull Emelyne M, Sendker Franziska, Bernhard Frank, Scheidt Holger A, Schmidt Peter, Huster Daniel, Krug Ulrike
Institute for Medical Physics and Biophysics, University of Leipzig, Leipzig, Germany.
Institute of Biophysical Chemistry, Goethe University Frankfurt, Frankfurt am Main, Germany.
Front Pharmacol. 2020 Oct 29;11:562113. doi: 10.3389/fphar.2020.562113. eCollection 2020.
Cell-free expression represents an attractive method to produce large quantities of selectively labeled protein for NMR applications. Here, cell-free expression was used to label specific regions of the growth hormone secretagogue receptor (GHSR) with NMR-active isotopes. The GHSR is a member of the class A family of G protein-coupled receptors. A cell-free expression system was established to produce the GHSR in the precipitated form. The solubilized receptor was refolded and reconstituted into DMPC lipid membranes. Methionines, arginines, and histidines were chosen for C-labeling as they are representative for the transmembrane domains, the loops and flanking regions of the transmembrane α-helices, and the C-terminus of the receptor, respectively. The dynamics of the isotopically labeled residues was characterized by solid-state NMR measuring motionally averaged H-C dipolar couplings, which were converted into molecular order parameters. Separated local field DIPSHIFT experiments under magic-angle spinning conditions using either varying cross polarization contact times or direct excitation provided order parameters for these residues showing that the C-terminus was the segment with the highest motional amplitude. The loop regions and helix ends as well as the transmembrane regions of the GHSR represent relatively rigid segments in the overall very flexible receptor molecule. Although no site resolution could be achieved in the experiments, the previously reported highly dynamic character of the receptor concluded from uniformly C labeled receptor samples could be further specified by this segmental labeling approach, leading to a more diversified understanding of the receptor dynamics under equilibrium conditions.
无细胞表达是一种颇具吸引力的方法，可用于生产大量用于核磁共振（NMR）应用的选择性标记蛋白质。在此，利用无细胞表达用具有NMR活性的同位素标记生长激素促分泌素受体（GHSR）的特定区域。GHSR是A类G蛋白偶联受体家族的成员。建立了一种无细胞表达系统，以沉淀形式生产GHSR。将溶解的受体重新折叠并重构到二肉豆蔻酰磷脂酰胆碱（DMPC）脂质膜中。选择甲硫氨酸、精氨酸和组氨酸进行¹³C标记，因为它们分别代表跨膜结构域、跨膜α螺旋的环和侧翼区域以及受体的C末端。通过固态NMR测量运动平均的H-C偶极耦合来表征同位素标记残基的动力学，该耦合被转换为分子序参数。在魔角旋转条件下使用不同的交叉极化接触时间或直接激发进行的分离局部场DIPSHIFT实验提供了这些残基的序参数，表明C末端是运动幅度最高的片段。GHSR的环区域、螺旋末端以及跨膜区域在整体非常灵活的受体分子中代表相对刚性的片段。尽管在实验中无法实现位点分辨率，但通过这种片段标记方法可以进一步明确先前从均匀¹³C标记的受体样品得出的受体高度动态特性，从而在平衡条件下对受体动力学有更全面的理解。
