Synthesis and biophysical analysis of transmembrane domains of a Saccharomyces cerevisiae G protein-coupled receptor.
Synthesis and biophysical analysis of transmembrane domains of a Saccharomyces cerevisiae G protein-coupled receptor.
复制标题
酿酒酵母 G 蛋白偶联受体跨膜结构域的合成和生物物理分析。
DOI：
10.1021/bi001432p
复制
发表时间：
2000
期刊：
影响因子：
2.9
通讯作者：
Naider,F
中科院分区：
文献类型：
--
作者：
Xie,H;Ding,FX;Schreiber,D;Eng,G;Liu,SF;Arshava,B;Arevalo,E;Becker,JM;Naider,F
α-因子的Ste 2 p受体是酿酒酵母（Saccharomycescerevisiae）的一种十三肽交配信息素，属于G蛋白偶联受体家族。在本文中，我们报告了与七个跨膜结构域（M1-M5）中的五个相对应的肽的合成，以及与野生型序列相对应的第六个跨膜结构域的两个同源物和在组成型活性受体中发现的突变序列。使用三氟乙醇、三氟乙醇-水混合物、十二烷基硫酸钠胶束和二肉豆蔻酰磷脂酰胆碱双层中的详细CD分析，评估了所有新的跨膜肽和先前合成的对应于结构域6和7的肽的二级结构。色氨酸荧光猝灭实验用于评估膜肽进入脂质双层的渗透。在三氟乙醇和大多数三氟乙醇-水混合物中，所有肽主要是（40 - 80%）螺旋。相比之下，两种肽M3-35（KKKNIIQVLLVASIETSLVIKVIFTGDNFKKKG）和M6-31（KQFDSFHILLINleSAQSLLVPSIIFILAYSLK）在十二烷基硫酸钠胶束和DMPC双层中都形成稳定的β-折叠结构。聚丙烯酰胺凝胶电泳表明，这两个肽形成高分子聚集体的存在下，SDS，而所有其他肽作为单体物种移动。与组成型突变体中的序列对应的肽（KKKFDSFHILLIMSAQSLLVLSIIFILAYSLKKKS）在各种条件下主要是螺旋的，而同源野生型序列（KKKFDSFHILLIMSAQSLLVPSIIFILAYSLKKKS）保留形成β结构的趋势。这些结果表明，二级结构中的构象转变，如通过生物物理技术检测到的，和受体功能之间的连接。特定跨膜结构域的聚集也可以反映在膜环境中发生的分子间相互作用的趋势，其促进受体二聚体或多聚体的形成。
The Ste2p receptor for α-factor, a tridecapeptide mating pheromone of the yeastSaccharomyces cerevisiae, belongs to the G protein-coupled family of receptors. In this paper we report on the synthesis of peptides corresponding to five of the seven transmembrane domains (M1−M5) and two homologues of the sixth transmembrane domain corresponding to the wild-type sequence and a mutant sequence found in a constitutively active receptor. The secondary structures of all new transmembrane peptides and previously synthesized peptides corresponding to domains 6 and 7 were assessed using a detailed CD analysis in trifluoroethanol, trifluoroethanol−water mixtures, sodium dodecyl sulfate micelles, and dimyristoyl phosphatidyl choline bilayers. Tryptophan fluorescence quenching experiments were used to assess the penetration of the membrane peptides into lipid bilayers. All peptides were predominantly (40−80%) helical in trifluoroethanol and most trifluoroethanol−water mixtures. In contrast, two of the peptides M3-35 (KKKNIIQVLLVASIETSLVFQIKVIFTGDNFKKKG) and M6-31 (KQFDSFHILLINleSAQSLLVPSIIFILAYSLK) formed stable β-sheet structures in both sodium dodecyl sulfate micelles and DMPC bilayers. Polyacrylamide gel electrophoresis showed that these two peptides formed high molecular aggregates in the presence of SDS whereas all other peptides moved as monomeric species. The peptide (KKKFDSFHILLIMSAQSLLVLSIIFILAYSLKKKS) corresponding to the sequence in the constitutive mutant was predominantly helical under a variety of conditions, whereas the homologous wild-type sequence (KKKFDSFHILLIMSAQSLLVPSIIFILAYSLKKKS) retained a tendency to form β-structures. These results demonstrate a connection between a conformational shift in secondary structure, as detected by biophysical techniques, and receptor function. The aggregation of particular transmembrane domains may also reflect a tendency for intermolecular interactions that occur in the membrane environment facilitating formation of receptor dimers or multimers.