All experiments were conducted at 25 °C in A25 buffer with 50 mM KCl and variable glycerol and nucleotide as indicated. Binding curves were fit to the full quadratic equation for tight binding (mutual depletion)
where P and X are the total concentrations and Y(bar) is the fraction of P with X bound. FRET from kinesin to mdADP (2′-deoxy-3′-mant-ADP) was measured using excitation at 280 nm and a 418 nm long pass filter for emission. mdADP was prepared by the method of Hiratsuka [20 (link)]. Stopped flow experiments were performed on a SX-20 instrument (Applied Photophysics). Fluorescence measurements in a stirred cuvet format were conducted in a custom designed thermostated chamber with fiber optics linking it to the SX-20 instrument that was used as the excitation light source and for data acquisition. ATPase rates were determined from the rate of decrease in absorbance of NADH at 340 nm using a coupled assay of pyruvate kinase and lactic dehydrogenase [21 ]. Nucleotides were added as the 1:1 complex with magnesium.
where P and X are the total concentrations and Y(bar) is the fraction of P with X bound. FRET from kinesin to mdADP (2′-deoxy-3′-mant-ADP) was measured using excitation at 280 nm and a 418 nm long pass filter for emission. mdADP was prepared by the method of Hiratsuka [20 (link)]. Stopped flow experiments were performed on a SX-20 instrument (Applied Photophysics). Fluorescence measurements in a stirred cuvet format were conducted in a custom designed thermostated chamber with fiber optics linking it to the SX-20 instrument that was used as the excitation light source and for data acquisition. ATPase rates were determined from the rate of decrease in absorbance of NADH at 340 nm using a coupled assay of pyruvate kinase and lactic dehydrogenase [21 ]. Nucleotides were added as the 1:1 complex with magnesium.