Iwamoto T, Grove A, Montal M O, Montal M, Tomich J M
Department of Biochemistry, Kansas State University, Manhattan.
Int J Pept Protein Res. 1994 Jun;43(6):597-607. doi: 10.1111/j.1399-3011.1994.tb00562.x.
A strategy for the synthesis of peptides and oligomeric proteins designed to form transmembrane ion channels is described. A folding motif that exhibits a functional ionic pore encompasses amphipathic alpha-helices organized as a four-helix bundle around a central hydrophilic pore. The channel-forming activity of monomeric amphipathic peptides may be examined after reconstitution in lipid bilayers in which peptides self-assemble into conductive oligomers. The covalent attachment of channel-forming peptides to the lysine epsilon-amino groups of a template molecule (KKKPGKEKG) specifies oligomeric number and facilitates the study of ionic permeation and channel blockade. Here we describe detailed protocols for the total synthesis of peptides and template-assembled four-helix bundle proteins, exemplified with the sequence of M2 delta (EKM-STAISVLLAQAVFLLLTSQR), considered involved in lining the pore of the nicotinic acetylcholine receptor channel. For comparison, the synthesis of a second four-helix bundle, T4CaIVS3 with the sequence of predicted transmembrane segment S3 (DPWNVFDFLIVIGSIIDVILSE) of the fourth repeat of the L-type voltage-gated calcium channel, is included. Peptides and proteins are synthesized step-wise by solid-phase methods, purified by reversed-phase HPLC, and homogeneity ascertained by analytical HPLC, capillary zone electrophoresis, SDS/PAGE, amino acid analysis and sequencing. Optimization of synthetic procedures for hydrophobic molecules include reducing resin substitution to avoid steric hindrance and aggregation of the final product. Protocols for the preparation of the samples prior to HPLC purification as well as the conditions and columns required for successful purification are presented. The methods developed are generally applicable for the chemical synthesis, purification and characterization of amphipathic peptides and template directed helical bundle proteins.
本文描述了一种合成旨在形成跨膜离子通道的肽和寡聚蛋白的策略。一种具有功能性离子孔的折叠基序包含两亲性α-螺旋，这些螺旋围绕中心亲水孔组织成四螺旋束。单体两亲性肽的通道形成活性可在重构于脂质双层后进行检测，在脂质双层中肽会自组装成导电寡聚体。将形成通道的肽共价连接到模板分子（KKKPGKEKG）的赖氨酸ε-氨基上可确定寡聚数，并有助于研究离子渗透和通道阻断。在此，我们描述了肽和模板组装的四螺旋束蛋白全合成的详细方案，以被认为参与烟碱型乙酰胆碱受体通道孔内衬的M2δ（EKM-STAISVLLAQAVFLLLTSQR）序列为例。作为比较，还包括第二个四螺旋束T4CaIVS3的合成，其序列为L型电压门控钙通道第四个重复序列的预测跨膜片段S3（DPWNVFDFLIVIGSIIDVILSE）。肽和蛋白通过固相方法逐步合成，通过反相高效液相色谱纯化，并通过分析型高效液相色谱、毛细管区带电泳、SDS/聚丙烯酰胺凝胶电泳、氨基酸分析和测序确定其均一性。疏水分子合成程序的优化包括降低树脂取代度以避免空间位阻和最终产物的聚集。本文还介绍了高效液相色谱纯化前样品制备的方案以及成功纯化所需的条件和柱子。所开发的方法一般适用于两亲性肽和模板导向螺旋束蛋白的化学合成、纯化和表征。
