Application Notes

2 Results We first analyzed the binding of an acceptor peptide to PglB. This acceptor peptide contains an asparagine residue within a consensus sequon, and it is fluorescently labeled with 5-Carboxyfluorescein (Table 1). The concentrations of labeled peptide and Mn 2+ , an essential PglB cofactor, were kept constant in all samples at 50 nM and 10 mM respectively, whilst the concentration of PglB was varied. The samples were incubated at 4 °C for 10 minutes, before loading into MST standard treated capillaries. The MST measurements were then performed at 10 °C. For the interaction of PglB with the acceptor peptide, a K d of 0.42 +/- 0.17 µM was determined (Figure 2). This value is in the same range of the K d value previously reported for the interaction of PglB with the same acceptor peptide. In that case the determination was performed using fluorescence anisotropy and the value was 1.02 +/- 0.06 µM [3]. Figure 2: Binding of the acceptor and inhibitor peptides to PgIB. Both 5-FAM fluorescently labeled peptides were used at a constant concentration of 50 nM with varying concentrations of PgB. The acceptor peptide binds with a K d of 0.42 +/- 0.17 µM to PglB (squares), while the binding affinity of the inhibitor peptide (triangles) to PglB was 3.80 +/- 0.20 µM. The error bars reflect standard deviation (SD) from 2 independent measurements. In a second experiment, we analyzed the binding of PglB to a peptide in which the asparagine residue within the glycosylation sequon, was replaced by 2,4-diaminobutanoic acid (Dab). This exchange has been shown to have inhibitory effects in some eukaryotic OST [4]. The inhibitory peptide, as well as the acceptor peptide, is labeled with 5-Carboxyfluorescein. The concentrations of labeled peptide and Mn 2+ were kept constant, while the concentration of PglB was varied. In this case, a K d of 3.80 +/- 0.2 µM was determined for the interaction between PglB and the inhibitory peptide. This value is again in the same range that the one previously reported for the same interaction, 10.15 +/- 0.25 µM. Furthermore, we observed that the affinity between PglB and the inhibitory peptide is ~ 10-fold lower than for the acceptor peptide, which is completely in concordance with our previous results, as shown in Table 1 (Lizak et al, 2013). The lower affinity observed for the interaction of PglB with the inhibitory peptide has been explained before by two different aspects: In the acceptor peptide the carbonyl of the asparagineresidue is likely interacting with PglB, whereas the positive charge of the primary amine group in the inhibitory peptide could be weakening the binding to PglB. Conclusions Our experiments show that MicroScale Themophoresis (MST) is a suitable tool for the analysis of interactions between detergent- solubilized membrane proteins and their substrates, as demonstrated here by analyzing the interaction of labeled peptides and the membrane enzyme PglB. The K d values that were determined for the interactions between PglB and the two different labeled peptides were in concordance with the values obtained using fluorescence anisotropy.

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