Figure 2

Alexa-calmodulin binding to Myo1c IQ peptides on plates A, Alexa-calmodulin (50 nM) was incubated in a 96-well plate covalently bound with Myo1c IQ peptides, a positive-control IQ peptide (NM), or a negative-control peptide (PVP); binding was carried out at room temperature in the presence of low (150 mM) or high (400 mM) KCl and in the presence of 100 μM EGTA. We measured unbound Alexa-calmodulin concentration by fluorescence, then inferred the amount bound. Averaged data from three independent experiments (error bars indicate standard error). B, competition with free IQ peptides. Following incubation of 50 nM Alexa-calmodulin and free IQ peptides in the presence of 100 μM EGTA and 150 mM KCl in wells of an IQ3-derivatized plastic plate, unbound Alexa-calmodulin was measured; error bars indicate standard deviation with n = 6 (single experiment). To correct for quenching of Alexa-calmodulin fluorescence by IQ peptides, each sample was standardized against an identical reaction in an underivatized plate. Data were fit with equation (4) with the Hill coefficient set at 2.