title: Expedient total synthesis of small to medium-sized membrane proteins via Fmoc chemistry.
abstract: Total chemical synthesis provides a unique approach for the access to uncontaminated, monodisperse, and more importantly, post-translationally modified membrane proteins. In the present study we report a practical procedure for expedient and cost-effective synthesis of small to medium-sized membrane proteins in multimilligram scale through the use of automated Fmoc chemistry. The key finding of our study is that after the attachment of a removable arginine-tagged backbone modification group, the membrane protein segments behave almost the same as ordinary water-soluble peptides in terms of Fmoc solid-phase synthesis, ligation, purification, and mass spectrometry characterization. The efficiency and practicality of the new method is demonstrated by the successful preparation of Ser64-phosphorylated M2 proton channel from influenza A virus and the membrane-embedded domain of an inward rectifier K(+) channel protein Kir5.1. Functional characterizations of these chemically synthesized membrane proteins indicate that they provide useful and otherwise-difficult-to-access materials for biochemistry and biophysics studies.
authors.affiliation: Tsinghua-Peking Center for Life Sciences, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University , Beijing 100084, China.
authors.forename: Ji-Shen
authors.initials: JS
authors.lastname: Zheng
authors.forename: Mu
authors.initials: M
authors.lastname: Yu
authors.forename: Yun-Kun
authors.initials: YK
authors.lastname: Qi
authors.forename: Shan
authors.initials: S
authors.lastname: Tang
authors.forename: Fei
authors.initials: F
authors.lastname: Shen
authors.forename: Zhi-Peng
authors.initials: ZP
authors.lastname: Wang
authors.forename: Liang
authors.initials: L
authors.lastname: Xiao
authors.forename: Longhua
authors.initials: L
authors.lastname: Zhang
authors.forename: Chang-Lin
authors.initials: CL
authors.lastname: Tian
authors.forename: Lei
authors.initials: L
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D005449:Fluorenes / Q000737:chemistry*; D007700:Kinetics; D008565:Membrane Proteins / Q000138:chemical synthesis* / Q000737:chemistry; D008969:Molecular Sequence Data; D010766:Phosphorylation; D024661:Potassium Channels, Inwardly Rectifying / Q000138:chemical synthesis / Q000737:chemistry; D017434:Protein Structure, Tertiary; D060327:Solid-Phase Synthesis Techniques / Q000379:methods*; D014269:Trifluoroacetic Acid / Q000737:chemistry; D014763:Viral Matrix Proteins / Q000138:chemical synthesis / Q000737:chemistry; D000097062:Kir5.1 Channel; D000086082:Viroporin Proteins
chemical_list: D005449:Fluorenes; D008565:Membrane Proteins; D024661:Potassium Channels, Inwardly Rectifying; D014269:Trifluoroacetic Acid; D014763:Viral Matrix Proteins; D000097062:Kir5.1 Channel; C496789:9-fluorenylmethoxycarbonyl; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins