Ahn Hee-Chul, Juranić Nenad, Macura Slobodan, Markley John L
National Magnetic Resonance Facility at Madison, Department of Biochemistry, University of Wisconsin-Madison, 53706-1544, USA.
J Am Chem Soc. 2006 Apr 5;128(13):4398-404. doi: 10.1021/ja057773d.
We chose crambin, a hydrophobic and water-insoluble protein originally isolated from the seeds of the plant Crambe abyssinica, as a model for NMR investigations of membrane-associated proteins. We produced isotopically labeled crambin(P22,L25) (variant of crambin containing Pro22 and Leu25) as a cleavable fusion with staphylococcal nuclease and refolded the protein by an approach that has proved successful for the production of proteins with multiple disulfide bonds. We used NMR spectroscopy to determine the three-dimensional structure of the protein in two membrane-mimetic environments: in a mixed aqueous-organic solvent (75%/25%, acetone/water) and in DPC micelles. With the sample in the mixed solvent, it was possible to determine (>NH...OC<) hydrogen bonds directly by the detection of (h3)J(NC)' couplings. H-bonds determined in this manner were utilized in the refinement of the NMR-derived protein structures. With the protein in DPC (dodecylphosphocholine) micelles, we used manganous ion as an aqueous paramagnetic probe to determine the surface of crambin that is shielded by the detergent. With the exception of the aqueous solvent exposed loop containing residues 20 and 21, the protein surface was protected by DPC. This suggests that the protein may be similarly embedded in physiological membranes. The strategy described here for the expression and structure determination of crambin should be applicable to structural and functional studies of membrane active toxins and small membrane proteins.
我们选择了胰凝乳蛋白酶原，一种最初从深黄美丁花植物种子中分离出来的疏水性且不溶于水的蛋白质，作为用于核磁共振研究膜相关蛋白的模型。我们制备了同位素标记的胰凝乳蛋白酶原（P22，L25）（含有Pro22和Leu25的胰凝乳蛋白酶原变体），使其与葡萄球菌核酸酶形成可裂解融合蛋白，并通过一种已被证明对生产具有多个二硫键的蛋白质成功的方法来复性该蛋白。我们使用核磁共振光谱法在两种模拟膜环境中确定该蛋白的三维结构：在混合水 - 有机溶剂（75%/25%，丙酮/水）中和在二癸基磷脂酰胆碱（DPC）胶束中。对于混合溶剂中的样品，通过检测（h3）J（NC）' 耦合可以直接确定（>NH...OC<）氢键。以这种方式确定的氢键被用于优化核磁共振衍生的蛋白质结构。对于处于DPC（十二烷基磷酸胆碱）胶束中的蛋白质，我们使用锰离子作为水性顺磁探针来确定被去污剂屏蔽的胰凝乳蛋白酶原的表面。除了包含残基20和21的暴露于水相溶剂的环外，蛋白质表面被DPC保护。这表明该蛋白质可能以类似方式嵌入生理膜中。这里描述的用于胰凝乳蛋白酶原表达和结构测定的策略应该适用于膜活性毒素和小膜蛋白的结构与功能研究。
