Figure 4: The Drg1-21 variant fails to dissociate from the substrate protein Rlp24.

Surface plasmon resonance measurements were performed on a BiacoreX system. GST-tagged Rlp24C was immobilized to CM5 sensor chips (GE Healthcare) and 100 nM of the respective Drg1 variants were injected as binding partners. The reference chamber contained empty GST. The association phase starts with the injection of the sample (0 seconds) and the dissociation phase begins at the end of the injection (120 seconds). Prior to injection, the Drg1 variants were pre-incubated with 1 mM ATP for 5 minutes. (a) Drg1 wildtype was purified in the absence of nucleotide and pre-incubated either with 1 mM ADP or 1 mM ATP. (b) Drg1 variants purified in the presence of 1 mM ADP were injected. All samples were incubated with 1 mM ATP prior to injection. The running buffer contained 1 mM ATP to analyse dissociation. (c) Drg1 variants purified in the absence of 1 mM ADP were injected. All samples were incubated with 1 mM ATP prior to injection. The running buffer contained 1 mM ATP to analyse dissociation. The displayed data are taken from representative experiments out of at least two biological replicates collected from two CM5 chips. (RU Response units).