Removable Backbone Modification Method for the Chemical Synthesis of Membrane Proteins.

Chemical synthesis can produce water-soluble globular proteins bearing specifically designed modifications. These synthetic molecules have been used to study the biological functions of proteins and to improve the pharmacological properties of protein drugs. However, the above advances notwithstanding, membrane proteins (MPs), which comprise 20-30% of all proteins in the proteomes of most eukaryotic cells, remain elusive with regard to chemical synthesis. This difficulty stems from the strong hydrophobic character of MPs, which can cause considerable handling issues during ligation, purification, and characterization steps. Considerable efforts have been made to improve the solubility of transmembrane peptides for chemical ligation. These methods can be classified into two main categories: the manipulation of external factors and chemical modification of the peptide. This Account summarizes our research advances in the development of chemical modification especially the two generations of removable backbone modification (RBM) strategy for the chemical synthesis of MPs. In the first RBM generation, we install a removable modification group at the backbone amide of Gly within the transmembrane peptides. In the second RBM generation, the RBM group can be installed into all primary amino acid residues. The second RBM strategy combines the activated intramolecular O-to-N acyl transfer reaction, in which a phenyl group remains unprotected during the coupling process, which can play a catalytic role to generate the activated phenyl ester to assist in the formation of amide. The key feature of the RBM group is its switchable stability in trifluoroacetic acid. The stability of these backbone amide N-modifications toward TFA can be modified by regulating the electronic effects of phenol groups. The free phenol group is acylated to survive the TFA deprotection step, while the acyl phenyl ester will be quantitatively hydrolyzed in a neutral aqueous solution, and the free phenol group increases the electron density of the benzene ring to make the RBM labile to TFA. The transmembrane peptide segment bearing RBM groups behaves like a water-soluble peptide during fluorenylmethyloxycarbonyl based solid-phase peptide synthesis (Fmoc SPPS), ligation, purification, and characterization. The quantitative removal of the RBM group can be performed to obtain full-length MPs. The RBM strategy was used to prepare the core transmembrane domain Kir5.1[64-179] not readily accessible by recombinant protein expression, the influenza A virus M2 proton channel with phosphorylation, the cation-specific ion channel p7 from the hepatitis C virus with site-specific NMR isotope labels, and so on. The RBM method enables the practical engineering of small- to medium-sized MPs or membrane protein domains to address fundamental questions in the biochemical, biophysical, and pharmaceutical sciences.

Expedient total synthesis of small to medium-sized membrane proteins via Fmoc chemistry.

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.