Buffy Jarrod J, Traaseth Nathaniel J, Mascioni Alessandro, Gor'kov Peter L, Chekmenev Eduard Y, Brey William W, Veglia Gianluigi
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Biochemistry. 2006 Sep 12;45(36):10939-46. doi: 10.1021/bi060728d.
Sarcolipin (SLN), a 31 amino acid integral membrane protein, regulates SERCA1a and SERCA2a, two isoforms of the sarco(endo)plasmic Ca-ATPase, by lowering their apparent Ca(2+) affinity and thereby enabling muscle relaxation. SLN is expressed in both fast-twitch and slow-twitch muscle fibers with significant expression levels also found in the cardiac muscle. SLN shares approximately 30% identity with the transmembrane domain of phospholamban (PLN), and recent solution NMR studies carried out in detergent micelles indicate that the two polypeptides bind to SERCA in a similar manner. Previous 1D solid-state NMR experiments on selectively (15)N-labeled sites showed that SLN crosses the lipid bilayer with an orientation nearly parallel to the bilayer normal. With a view toward the characterization of SLN structure and its interactions with both lipids and SERCA, herein we report our initial structural and topological assignments of SLN in mechanically oriented DOPC/DOPE lipid bilayers as mapped by 2D (15)N PISEMA experiments. The PISEMA spectra obtained on uniformly (15)N-labeled protein as well as (15)N-Leu, (15)N-Ile and (15)N-Val map the secondary structure of SLN and, simultaneously, reveal that SLN exists in two distinct topologies. Both the major and the minor populations assume an orientation with the helix axis tilted by approximately 23 degrees with respect to the lipid bilayer normal, but vary in the rotation angle about the helix axis by approximately 5 degrees . The existence of the multiple populations in model membranes may be a significant requirement for SLN interaction with SERCA.
肌浆球蛋白（SLN）是一种由31个氨基酸组成的整合膜蛋白，它通过降低肌浆（内质）网Ca-ATP酶的两种同工型SERCA1a和SERCA2a的表观Ca(2+)亲和力来调节它们，从而实现肌肉松弛。SLN在快肌纤维和慢肌纤维中均有表达，在心肌中也有显著的表达水平。SLN与受磷蛋白（PLN）的跨膜结构域具有约30%的同源性，最近在去污剂胶束中进行的溶液核磁共振研究表明，这两种多肽以相似的方式与SERCA结合。先前对选择性（15）N标记位点进行的一维固态核磁共振实验表明，SLN以几乎平行于双层法线的方向穿过脂质双层。为了表征SLN的结构及其与脂质和SERCA的相互作用，在此我们报告通过二维（15）N PISEMA实验绘制的在机械取向的DOPC/DOPE脂质双层中SLN的初始结构和拓扑归属。在均匀（15）N标记的蛋白质以及（15）N-亮氨酸、（15）N-异亮氨酸和（15）N-缬氨酸上获得的PISEMA光谱描绘了SLN的二级结构，同时揭示SLN存在两种不同的拓扑结构。主要群体和次要群体的螺旋轴相对于脂质双层法线倾斜约23度，但绕螺旋轴的旋转角度相差约5度。模型膜中多种群体的存在可能是SLN与SERCA相互作用的一个重要条件。
