Expedient Total Synthesis of Small to Medium-Sized Membrane Proteins via Fmoc Chemistry

• Published in: Journal of the American Chemical Society Vol. 136; no. 9; pp. 3695 - 3704
• Main Authors: Zheng, Ji-Shen, Yu, Mu, Qi, Yun-Kun, Tang, Shan, Shen, Fei, Wang, Zhi-Peng, Xiao, Liang, Zhang, Longhua, Tian, Chang-Lin, Liu, Lei
• Format: Journal Article
• Language: English
• Published: WASHINGTON American Chemical Society 05.03.2014; Amer Chemical Soc
• Subjects: Amino Acid Sequence; biophysics; Chemistry; Chemistry, Multidisciplinary; cost effectiveness; Fluorenes - chemistry; Influenza A virus; Kinetics; Kir5.1 Channel; mass spectrometry; Membrane Proteins - chemical synthesis; Membrane Proteins - chemistry; Molecular Sequence Data; peptides; Phosphorylation; Physical Sciences; potassium channels; Potassium Channels, Inwardly Rectifying - chemical synthesis; Potassium Channels, Inwardly Rectifying - chemistry; Protein Structure, Tertiary; Science & Technology; Solid-Phase Synthesis Techniques - methods; synthesis; Trifluoroacetic Acid - chemistry; Viral Matrix Proteins - chemical synthesis; Viral Matrix Proteins - chemistry; TOTAL CHEMICAL-SYNTHESIS; DOMAIN; SEMISYNTHESIS; CHANNEL; MECHANISM; ESCHERICHIA-COLI; LIGATION; VIRUS M2 PROTEIN; AMIDE BOND; PEPTIDE
• DOI: 10.1021/ja500222u
• ISSN: 0002-7863; 1520-5126

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.