To allow more precise determination of the differences in the affinities of Rho3GV, Rho3EV, and wild-type Rho3, we performed binding experiments with a lower concentration of ligands (100 µg/ml).
Because weaker interactions with high micromolar affinities, such as serotonin at wild Ac-AChBP, are difficult to measure with a radioligand binding assay, we verified our binding experiments with a surface plasmon resonance (SPR) biosensor assay [ 14] (Table 1).
This proposed 'twisted amide' activation of the acceptor asparagine has gained further support with kinetic measurements and peptide binding experiments with a range of acceptor analogues and with mutated PglB [ 11].
We therefore derivatised one of the more soluble compounds (1acc) with a fluorescein label to give FITC-Gly-1acc and performed a direct binding experiment with hDM2 this afforded a dissociation constant of 760 (±140) nM and confirmed strong interaction with the target protein.
These structural observations are consistent with RNA binding experiments with all possible single substitutions in 3′ end nucleotides 1 11, which show that nucleotides 6 8 are the most critical for sequence specific binding.
Three days prior to a binding experiment with LigandTracer®, petri dishes with approximately 106 cells were prepared (Fig. 1).
The binding experiment with hnRNPH1 showed that there is a correlation between the binding of hnRNPH1 to a certain pri-miRNA and the contribution of this protein to miRNA processing, as implicated from the siRNA experiment.
As for binding affinity, we have included a competitive binding experiment with radiolabeled DHT in cells expressing the WT or mutant receptor, showing increased affinity of enzalutamide for F876L (comparable to DHT affinity), consistent with conversion to agonism (new figure: Figure 2– figure supplement 3).
For each binding experiment with one substrate, 1 mM solution of the ligand was introduced from the syringe into the cell containing the enzyme saturated with the other substrate.
The binding experiment with the biotinylated soy bean trypsin inhibitor was done similarly, except the detection was made using 1∶4000 dilution of streptavidin-HRP (Sigma, S2438) according to the manufactures instructions.
