Department of Chemistry and Biochemistry, University of California, Santa Barbara, Santa Barbara, California, USA.
Biophys J. 2013 Jan 22;104(2):472-81. doi: 10.1016/j.bpj.2012.11.3831.
Oligomerization has important functional implications for many membrane proteins. However, obtaining structural insight into oligomeric assemblies is challenging, as they are large and resist crystallization. We focus on proteorhodopsin (PR), a protein with seven transmembrane α-helices that was found to assemble to hexamers in densely packed lipid membrane, or detergent-solubilized environments. Yet, the structural organization and the subunit interface of these PR oligomers were unknown. We used site-directed spin-labeling together with electron spin-resonance lineshape and Overhauser dynamic nuclear polarization analysis to construct a model for the specific orientation of PR subunits within the hexameric complex. We found intersubunit distances to average 16 Å between neighboring 55 residues and that residues 177 are >20 Å apart from each other. These distance constraints show that PR has a defined and radial orientation within a hexamer, with the 55-site of the A-B loop facing the hexamer core and the 177-site of the E-F loop facing the hexamer exterior. Dynamic nuclear polarization measurements of the local solvent dynamics complement the electron spin-resonance-based distance analysis, by resolving whether protein surfaces at positions 55, 58, and 177 are exposed to solvent, or covered by protein-protein or protein-detergent contacts.
寡聚化对许多膜蛋白具有重要的功能意义。然而，获得寡聚体结构的深入了解具有挑战性，因为它们很大，难以结晶。我们专注于视紫红质（PR），这是一种具有七个跨膜α螺旋的蛋白质，在密集堆积的脂质膜或去污剂溶解的环境中被发现组装成六聚体。然而，这些 PR 寡聚体的结构组织和亚基界面尚不清楚。我们使用定点自旋标记，结合电子自旋共振线宽和 Overhauser 动态核极化分析，构建了 PR 亚基在六聚体复合物中特定取向的模型。我们发现，相邻 55 个残基之间的亚基间距离平均为 16 Å，而残基 177 彼此之间的距离>20 Å。这些距离约束表明 PR 在六聚体中具有确定的和径向的取向，A-B 环的 55 位朝向六聚体核心，E-F 环的 177 位朝向六聚体外部。局部溶剂动力学的动态核极化测量补充了基于电子自旋共振的距离分析，通过确定位置 55、58 和 177 的蛋白质表面是否暴露于溶剂，或被蛋白质-蛋白质或蛋白质-去污剂接触覆盖。
