Naider Fred, Estephan Racha, Englander Jacqueline, Suresh Babu V V, Arevalo Enrique, Samples Karen, Becker Jeffrey M
Department of Chemistry, The College of Staten Island of The City University of New York, Staten Island, NY 10314, USA.
Biopolymers. 2004;76(2):119-28. doi: 10.1002/bip.10567.
The yeast Saccharomyces cerevisiae undergoes cell fusion during sexual conjugation to form diploid cells. The haploids participating in this process signal each other through secreted peptide-mating factors (alpha-factor and a-factor) that are recognized by G-protein-coupled receptors. The receptor (Ste2p) recognizing the tridecapeptide alpha-factor is used as a model system in our laboratory to understand various aspects of peptide-receptor interactions and receptor structure. Using chemical procedures we have synthesized peptides corresponding to the seven transmembrane domains of Ste2p and studied their structures in membrane mimetic environments. Extension of these studies requires preparation of longer fragments of Ste2p. This article discusses strategies used in our laboratory to prepare peptides containing multiple domains of Ste2p. Data are presented on the use of chemical synthesis, biosynthesis, and native chemical ligation. Using biosynthetic approaches fusion proteins have been expressed that contain single receptor domains, two transmembrane domains connected by the contiguous loop, and the tail connected to the seventh transmembrane domain. Tens of milligrams of fusion protein were obtained per liter, and multimilligram quantities of the isotopically labeled target peptides were isolated using such biosynthetic approaches. Initial circular dichroism results on a chemically synthesized 64-residue peptide containing a portion of the cytosolic tail and the complete seventh transmembrane domain showed that the tail portion and the hydrophobic core of this peptide maintained individual conformational preferences. Moreover, this peptide could be studied at near millimolar concentrations in the presence of micelles and did not aggregate under these conditions. Thus, these constructs can be investigated using high-resolution nuclear magnetic resonance techniques, and the cytosolic tail of Ste2p can be used as a hydrophilic template to improve solubility of transmembrane peptides for structural analysis.
酿酒酵母在有性接合过程中会发生细胞融合以形成二倍体细胞。参与此过程的单倍体通过分泌的肽类交配因子（α因子和a因子）相互发出信号，这些因子可被G蛋白偶联受体识别。在我们实验室中，识别十三肽α因子的受体（Ste2p）被用作模型系统，以了解肽-受体相互作用和受体结构的各个方面。我们使用化学方法合成了与Ste2p的七个跨膜结构域相对应的肽，并在膜模拟环境中研究了它们的结构。要扩展这些研究，需要制备更长的Ste2p片段。本文讨论了我们实验室中用于制备包含Ste2p多个结构域的肽的策略。文中展示了化学合成、生物合成和天然化学连接的应用数据。利用生物合成方法表达了融合蛋白，这些融合蛋白包含单个受体结构域、由连续环连接的两个跨膜结构域以及与第七个跨膜结构域相连的尾部。每升可获得数十毫克的融合蛋白，并且使用这种生物合成方法可分离出数毫克的同位素标记目标肽。对一个包含部分胞质尾部和完整第七个跨膜结构域的64个残基化学合成肽进行的初始圆二色性结果表明，该肽的尾部和疏水核心保持各自的构象偏好。此外，在存在胶束的情况下，可以在近毫摩尔浓度下研究该肽，并且在这些条件下它不会聚集。因此，可以使用高分辨率核磁共振技术研究这些构建体，并且Ste2p的胞质尾部可以用作亲水模板来提高跨膜肽的溶解度以进行结构分析。
