Chemical Synthesis of Native S‐Palmitoylated Membrane Proteins through Removable‐Backbone‐Modification‐Assisted Ser/Thr Ligation

Abstract

The preparation of native S‐palmitoylated (S‐palm) membrane proteins is one of the unsolved challenges in chemical protein synthesis. Herein, we report the first chemical synthesis of S‐palm membrane proteins by removable‐backbone‐modification‐assisted Ser/Thr ligation (RBMGABA‐assisted STL). This method involves two critical steps: 1) synthesis of S‐palm peptides by a new γ‐aminobutyric acid based RBM (RBMGABA) strategy, and 2) ligation of the S‐palm RBM‐modified peptides to give the desired S‐palm product by the STL method. The utility of the RBMGABA‐assisted STL method was demonstrated by the synthesis of rabbit S‐palm sarcolipin (SLN) and S‐palm matrix‐2 (M2) ion channel. The synthesis of S‐palm membrane proteins highlights the importance of developing non‐NCL methods for chemical protein synthesis.

Site-specific lipidation enhances IFITM3 membrane interactions and antiviral activity

“…Furthermore, a flotation assay revealed the disordered and amphipathic regions of Cys72-mPalm IFITM3 had increased association with the membrane bilayer. We note that maleimide-palmitate modified IFITM3 does not contain the native thioester linkage, which could be addressed through biophysical studies of site-specifically S-palmitoylated IFITM3 from new protein semisynthesis methods (Harmand, Pattabiraman, & Bode, 2017;D. L. Huang et al, 2020).…”

Total Synthesis and Structural Characterization of Caveolin‐1

“…[12][13][14][15][16][17][18] The synthesis of monopalmitoylated proteins has also been reported. [19][20][21] However, the establishment of the general synthetic route for the hydrophobic proteins including membrane proteins is still required to promote the research of these proteins. We previously succeeded in the synthesis of hydrophobic proteins with homogeneous glycan chains [22,23] by incorporating the Oacyl isopeptide method, [24] which is efficient to increase the solubility of peptide segments.…”

Sarcolipin alters SERCA1a interdomain communication by impairing binding of both calcium and ATP

“…To investigate a possible role of palmitoylation on SERCA1a activity, pSLN and SLN were chemically synthesized by a Ser/Thr ligation (STL) method (Fig. 2a) following the protocol we designed recently 29 . In brief, taking benefit of the presence of Thr13, two peptides (peptides 1 and 2, Fig.…”

“…in the absence of endogenous SLN, following the procedure we developed a few years ago 30 . Rabbit palmitoylated and unacylated SLN were chemically synthesized in accordance with our recent procedure 29 . As reconstitution protocols do not always allow an accurate control of protein orientation, we choose to work with solubilized forms as detailed in the Supplementary information, assuming that mixing of solubilized SLN (palmitoylated or not) with solubilized SERCA1a will allow the two proteins to interact properly as previously shown by NMR in the presence of dodecylphosphocholine 26 .…”

“…In the present study, we used chemically synthesized S-palmitoylated and non-palmitoylated rabbit SLN (dubbed "pSLN" and "SLN", respectively) 29 , as well as recombinant rabbit SERCA1a produced in the yeast S. cerevisiae 30 to investigate the regulatory role of SLN on SERCA1a. Therefore, in our experimental set-up, the two proteins are (i) from the same species, i.e.…”