Department of Chemistry, University of Toronto Mississauga, Mississauga, ON L5L 1C6, Canada.
J Biomol NMR. 2011 Sep;51(1-2):173-83. doi: 10.1007/s10858-011-9551-z. Epub 2011 Sep 27.
In studies of membrane proteins, knowledge of protein topology can provide useful insight into both structure and function. In this work, we present a solution NMR method for the measurement the tilt angle and average immersion depth of alpha helices in membrane proteins, from analysis of the paramagnetic relaxation rate enhancements arising from dissolved oxygen. No modification to the micelle or protein is necessary, and the topology of both transmembrane and amphipathic helices are readily determined. We apply this method to the measure the topology of a monomeric mutant of phospholamban (AFA-PLN), a 52-residue membrane protein containing both an amphipathic and a transmembrane alpha helix. In dodecylphosphocholine micelles, the amphipathic helix of AFA-PLN was found to have a tilt angle of 87° ± 1° and an average immersion depth of 13.2 Å. The transmembrane helix was found to have an average immersion depth of 5.4 Å, indicating residues 41 and 42 are closest to the micelle centre. The resolution of paramagnetic relaxation rate enhancements from dissolved oxygen compares favourably to those from Ni (II), a hydrophilic paramagnetic species.
在膜蛋白的研究中，蛋白质拓扑结构的知识可以为结构和功能提供有用的见解。在这项工作中，我们提出了一种基于溶液 NMR 的方法，用于测量膜蛋白中α螺旋的倾斜角和平均浸入深度，方法是分析溶解氧引起的顺磁弛豫率增强。不需要对胶束或蛋白质进行修饰，并且很容易确定跨膜和两亲性螺旋的拓扑结构。我们将该方法应用于测定磷酸化酶抑制蛋白（AFA-PLN）单体突变体的拓扑结构，该蛋白是一种 52 个残基的膜蛋白，含有一个两亲性和一个跨膜α螺旋。在十二烷基磷酸胆碱胶束中，AFA-PLN 的两亲性螺旋的倾斜角为 87°±1°，平均浸入深度为 13.2 Å。跨膜螺旋的平均浸入深度为 5.4 Å，表明残基 41 和 42 最接近胶束中心。溶解氧引起的顺磁弛豫率增强的分辨率与亲水性顺磁物质 Ni（II）相当。
