Abstract
Native chemical ligation (NCL) approaches have been applied extensively to soluble proteins. Fewer successes have been achieved with membrane peptides. In this report, the synthesis and semisynthesis by NCL of peptides corresponding to 1.7 transmembrane domains of the α-factor receptor from Saccharomyces cerevisiae is described. Synthesis was achieved when the ligation point was approximately in the middle of the loop joining the two transmembrane regions. In contrast, little to no ligation was observed when the ligation point was at the putative membrane interface of the sixth transmembrane domain (TM6) and the third extracellular loop (EL3). Ligations of a chemically synthesized 22-residue thioester with a synthetic 29-residue N-Cys peptide and a biosynthetic 73-residue N-Cys peptide were successfully achieved in both trifluoroethanol/guanidinium hydrochloride (TFE/GnHCl) and sodium dodecyl sulfate (SDS) media when mercaptoethanesulfonic acid (MESNA) was used as a catalyst. The resulting 51-residue and 95-residue ligation products were purified by reversed phase HPLC and recovered on a mg scale. Both peptides were >95% pure as determined by HPLC and had the expected molecular weight as judged by mass spectrometry. Segmental labeling of the 95-residue fragment, in which the N-Cys portion was [15N] labeled, resulted in a peptide that gave an NMR spectrum which was comparable to that of the unligated 73-residue peptide alone.
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Support was provided by National Institutes of Health Grants GM22086 and GM22087.
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R B Merrifield personified the finest qualities of a human being. He was an outstanding individual who influenced the way research is conducted by tens of thousands of scientists. At the same time he was a warm, humble, sincere man who was extremely kind and generous. I (FN) personally saw his generosity during a seminar he invited me to give at Rockefeller University. He was already a Nobel laureate but he treated me as a colleague and the encouragement he offered concerning my research program was very important for my future in academia. It is an honor to be among the participants in a volume honoring his contributions to peptide science.
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Balambika, R., Inui, T., Sargsyan, H. et al. Synthesis of a Double Transmembrane Domain Fragment of Ste2p by Native Chemical Ligation. Int J Pept Res Ther 13, 251–263 (2007). https://doi.org/10.1007/s10989-006-9076-6
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DOI: https://doi.org/10.1007/s10989-006-9076-6