Debelyy Mykhaylo O, Waridel Patrice, Quadroni Manfredo, Schneiter Roger, Conzelmann Andreas
Division of Biochemistry, Department of Biology, University of Fribourg, Fribourg, Switzerland.
Protein Analysis Facility, Center of Integrative Genomics, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland.
PLoS One. 2017 Oct 26;12(10):e0186840. doi: 10.1371/journal.pone.0186840. eCollection 2017.
Here we made an attempt to obtain partial structural information on the topology of multispan integral membrane proteins of yeast by isolating organellar membranes, removing peripheral membrane proteins at pH 11.5 and introducing chemical crosslinks between vicinal amino acids either using homo- or hetero-bifunctional crosslinkers. Proteins were digested with specific proteases and the products analysed by mass spectrometry. Dedicated software tools were used together with filtering steps optimized to remove false positive crosslinks. In proteins of known structure, crosslinks were found only between loops residing on the same side of the membrane. As may be expected, crosslinks were mainly found in very abundant proteins. Our approach seems to hold to promise to yield low resolution topological information for naturally very abundant or strongly overexpressed proteins with relatively little effort. Here, we report novel XL-MS-based topology data for 17 integral membrane proteins (Akr1p, Fks1p, Gas1p, Ggc1p, Gpt2p, Ifa38p, Ist2p, Lag1p, Pet9p, Pma1p, Por1p, Sct1p, Sec61p, Slc1p, Spf1p, Vph1p, Ybt1p).
在这里，我们尝试通过分离细胞器膜、在pH 11.5下去除外周膜蛋白，并使用同型或异型双功能交联剂在相邻氨基酸之间引入化学交联，来获取酵母多跨膜整合蛋白拓扑结构的部分信息。用特定蛋白酶消化蛋白质，并通过质谱分析产物。使用专用软件工具以及经过优化以去除假阳性交联的过滤步骤。在已知结构的蛋白质中，仅在膜同一侧的环之间发现交联。正如预期的那样，交联主要存在于非常丰富的蛋白质中。我们的方法似乎有望以相对较少的努力为天然非常丰富或强烈过表达的蛋白质提供低分辨率的拓扑信息。在此，我们报告了17种整合膜蛋白（Akr1p、Fks1p、Gas1p、Ggc1p、Gpt2p、Ifa38p、Ist2p、Lag1p、Pet9p、Pma1p、Por1p、Sct1p、Sec61p、Slc1p、Spf1p、Vph1p、Ybt1p）基于交联质谱的新拓扑数据。
