Application Notes

Studying the interaction of membrane enzyme PgIB with substrate and inhibitory peptide

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1 Membrane Proteins Application Note NT-MO-024 Studying the interaction of membrane enzyme PgIB with substrate and inhibitory peptide Ana Ramirez 1 , Moran Jerabek-Willemsen 2 , Heide Marie Roth 2 , Kaspar P. Locher 1 1 Institute of Molecular Biology and Biophysics, ETH Zurich, Zurich, Switzerland 2 NanoTemper Technologies GmbH, Munich, Germany Abstract During the catalytic cycle of enzymatic reactions, the recognition of a specific substrate by the enzyme is one of the most important steps, and the analysis of the affinity between enzyme and substrates and/or inhibitors is an important aspect in mechanistic biochemistry. Here, we have performed an analysis of the binding affinity between two different peptides and the bacterial oligosaccharyltransferase, PglB. Using labeled peptides, we have performed MicroScale Themophoresis experiments to determine the K d values for the interaction between PglB and these peptides. The results show a high concordance with the values previously reported for the same interaction determined by fluorescence anisotropy, showing that MST is a suitable tool for the study of membrane protein-substrate interactions in detergent solutions. Introduction N-glycosylation is a protein modification that occurs in all three kingdoms of life. It consists of the transfer of a glycan from a lipid-linked carrier to an asparagine residue within a glycosylation sequon (D/E-x-N-X-S/T for bacteria, N-X-S/T for archaea and eukaryotes) [1]. In bacteria, the reaction is catalyzed by the oligosaccharyl- transferase, PglB. Recently, the crystal structure of PglB was solved [2], and various studies at functional level have allowed a better understanding of the mechanism of N-glycosylation (Figure 1). One of the most important aspects that have been studied is the interaction of PglB with the acceptor peptide. Recently, it was reported how modifications in the asparagine that is glycosylated by PglB, have an influence in the peptide binding and activity of PglB [3]. In those studies, the K d values for the interactions of PglB with different peptides were determined by fluorescence anisotropy, using synthetic, labeled peptides. Here, we show the use of MicroScale Themophoresis (MST) to analyze the interaction of two different labeled peptides with PglB. One of them containing the wild type version of the glycosylation sequon, while in the other peptide the asparagine residue of the sequon has been replaced by 2,4-diaminobutanoic acid. Figure 1. Reaction cycle of N-glycosylation by PgIB. PgIB first binds to a substrate peptide. In the next step, a glycan from a lipid-linked carrier is engaged and transfered to an asparagine residue in the peptide. The glycosylated peptide is released from PgIB which is then free to start a new reaction cycle.

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