Abstract
Chemical synthesis can produce membrane proteins bearing specifically designed modifications (e.g., phosphorylation, isotope labeling) that are difficult to obtain through recombinant protein expression approaches. The resulting homogeneously modified synthetic membrane proteins are valuable tools for many advanced biochemical and biophysical studies. This protocol describes the chemical synthesis of membrane proteins by condensation of transmembrane peptide segments through native chemical ligation. To avoid common problems encountered due to the poor solubility of transmembrane peptides in almost any solvent, we describe an effective procedure for the chemical synthesis of membrane proteins through the removable-backbone modification (RBM) strategy. Two key steps of this protocol are: (i) installation of solubilizing Arg4-tagged RBM groups into the transmembrane peptides at any primary amino acid through Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis and (ii) native ligation of the full-length sequence, followed by removal of the RBM tags by TFA (trifluoroacetic acid) cocktails to afford the native protein. The installation of RBM groups is achieved by using 4-methoxy-5-nitrosalicyladehyde by reduction amination to incorporate an activated O-to-N acyl transfer auxiliary. The Arg4-tag-modified membrane-spanning peptide segments behave like water-soluble peptides to facilitate their purification, ligation and mass characterization.
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Acknowledgements
This study was supported by the National Key R&D Program of China (no. 2017YFA0505200) and the National Natural Science Foundation of China (grant nos. 21532004, 81621002, 21621003 and 21402206).
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L.L., J.-S.Z. and C.-L.T. conceived and led the project and wrote the manuscript. S.T., C.Z., C.-L.T., X.-Y.C., D.-L.H. and L.-H.Z. conducted the experiments.
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Integrated supplementary information
Supplementary Figure 1 Synthesis of M2[1-49, A30, RBM]-NHNH2.
(a) RP-HPLC analysis of the crude peptide M2[1-49, A30, RBM-Ac]-NHNH2 1' running on a C4- column. The linear gradient for analytical HPLC: 5% buffer B in buffer A to 95% B in A over 30 min. (b) ESI-MS of 1'. (c) ESI-MS of M2[1-49, A30, RBM]-NHNH2 1. (c) RP-HPLC analysis of the purified peptide M2[1-49, A30, RBM]-NHNH2 1 running on a C4- column. The linear gradient for analytical HPLC: 5% buffer B in buffer A to 95% B in A over 30 min. (d) ESI-MS of 1.
Supplementary Figure 2 Synthesis of M2[50-97, pSer64] (2).
(a) RP-HPLC analysis of the crude peptide 2 running on a C4- column. The linear gradient for analytical HPLC: 5% buffer B in buffer A to 95% B in A over 30 min. (b) RP-HPLC analysis of the purified peptide 2 running on a C4-column. The linear gradient for analytical HPLC: 20% buffer B in buffer A for 10 min, and then 20% buffer B in buffer A to 95% B in A over 30 min. (c) ESI-MS of 2.
Supplementary Figure 3 ESI-MS of 1′′ and 3.
(a) ESI-MS of 1”. (b) ESI-MS of 3.
Supplementary Figure 4 ESI-MS of 4.
Insert figure caption here by deleting or overwriting this text; captions may run to a second page if necessary.
Supplementary Figure 5 Synthesis of M2-TM(22-46).
(a) ESI-MS of M2-TM(22-46,RBM-Ac). (b) RP-HPLC analysis of the crude peptide of M2-TM(22-46,RBM) running on a C4- column. (c) RP-HPLC analysis of the purified peptide M2-TM(22-46,RBM) running on a C4-column. The linear gradient for analytical HPLC: 20% buffer B in buffer A for 10 min, and then 20% buffer B in buffer A to 95% B in A over 30 min. (d) ESI-MS of M2-TM(22-46,RBM). (e) RP-HPLC analysis of the purified peptide M2-TM(22-46) running on a C4-column. The linear gradient for analytical HPLC: 20% buffer B in buffer A to 95% B in A over 30 min. (f) ESI-MS of M2-TM(22-46).
Supplementary Figure 6 Synthesis of native M2.
(a) RP-HPLC analysis of native M2 running on a C4-column. The linear gradient for analytical HPLC: 40% buffer B in buffer A to 95% B in A over 30 min. (b) ESI-MS of native M2.
Supplementary Figure 7 Circular dichroism spectroscopy of purified M2, M2-TM(22-46) and M2-pSer64.
~10 μM of M2, M2-TM(22-46) or M2-pSer64 in 20 mM Tris, 50 mM OG,50 mM NaCl (pH 7.3) is analyzed in a 1 mm quartz cell.
Supplementary Figure 8 M2-pSer64-liposome-based fluorescent dye permeability assay.
Small light yellow circles represent 5(6)-carboxyfluorescein (CF) at self-quenching concentrations, and the small dark yellow circles represent CF being released from the liposomes (large black circles) after the addition of M2-pSer64, M2, and M2-TM (22-46). 0.5% Triton-X 100 was used to break the liposome and completely released the CF. The background fluorescence was measured with 2.5 % TFE added to liposome suspension.
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Tang, S., Zuo, C., Huang, DL. et al. Chemical synthesis of membrane proteins by the removable backbone modification method. Nat Protoc 12, 2554–2569 (2017). https://doi.org/10.1038/nprot.2017.129
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DOI: https://doi.org/10.1038/nprot.2017.129
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