Robust Chemical Synthesis of Membrane Proteins through a General Method of Removable Backbone Modification

Abstract

Chemical protein synthesis can provide access to proteins with post-translational modifications or site-specific labelings. Although this technology is finding increasing applications in the studies of water-soluble globular proteins, chemical synthesis of membrane proteins remains elusive. In this report, a general and robust removable backbone modification (RBM) method is developed for the chemical synthesis of membrane proteins. This method uses an activated O-to-N acyl transfer auxiliary to install in the Fmoc solid-phase peptide synthesis process a RBM group with switchable reactivity toward trifluoroacetic acid. The method can be applied to versatile membrane proteins because the RBM group can be placed at any primary amino acid. With RBM, the membrane proteins and their segments behave almost as if they were water-soluble peptides and can be easily handled in the process of ligation, purification, and mass characterizations. After the full-length protein is assembled, the RBM group can be readily removed by trifluoroacetic acid. The efficiency and usefulness of the new method has been demonstrated by the successful synthesis of a two-transmembrane-domain protein (HCV p7 ion channel) with site-specific isotopic labeling and a four-transmembrane-domain protein (multidrug resistance transporter EmrE). This method enables practical synthesis of small- to medium-sized membrane proteins or membrane protein domains for biochemical and biophysical studies.

A Helping Hand to Overcome Solubility Challenges in Chemical Protein Synthesis

“…46,47 Building on this work, Liu’s group recently published a method to introduce removable backbone modifications (RBMs) via a salicylaldehyde derivative. 48 …”

Total Chemical Synthesis of ISGylated-Ubiquitin Hybrid Chain Assisted by Acetamidomethyl Derivatives with Dual Functions

“…Similar phenomena were reported in cases of some highly hydrophobic proteins. 29,30 The isolated final protein was analyzed on SDS−PAGE with the Ub fragment, 6, and the full ISG15-thioester, 5b ( Figure 2F). The SDS gel showed that the isolated final protein has a mass that ranged between 25 and 35 kDa, which corresponds to our final ISG15-Ub hybrid chain molecular weight.…”

The Application of Isoacyl Structural Motifs in Prodrug Design and Peptide Chemistry

“…In addition to overcoming steric hindrance that resulted in different coupling steps, backbone amide substitution also effectively disrupted peptide aggregation by weakening the interchain hydrogen‐bonding network. This strategy was further developed by the groups of Johnson, Offer, and Liu . It is worth noting that the group of Liu extended the approach by introducing a solubilizing tag onto the auxiliary benzyl group that empowered the synthesis and handling of small to medium‐sized membrane proteins with multiple transmembrane domains (Scheme C).…”

“…This strategy was further developed by the groups of Johnson, [59] Offer, [60] and Liu. [61] It is worth noting that the group of Liu extended the approach by introducing as olubilizing tag onto the auxiliary benzyl group that empowered the synthesis and handling of small to medium-sized membrane proteins with multiple transmembrane domains (Scheme 4C). The tag was attached after completion of the target peptide assembly and anchored at the aniline amine, which was newly generated by the reduction of the nitro group.…”