Figure 3.

(A) Fluorescence measurement of the time course of association of NusDHFR with, and subsequent dissociation of the protein from, lipid vesicles containing MTX-PE. To fluorescein-NusDHFR (1 μg/mL) in a stirred cuvette, POPC/MTX-PE/RhoPE vesicles (95:4:1 molar proportions, 21 μM for the sample trace shown) were added at the time indicated by the first arrow. At the time indicated by the second arrow, 15 μM free MTX was added. The dashed line indicates the level to which the protein fluorescence falls due to inner-filter effects immediately upon vesicle addition. (B) Fit of the decrease in fluorescence following vesicle addition in panel A (omitting the instantaneous decrease due to inner-filter effects) to an exponentially falling function with rate constant k_Q. (Inset) Fit of the increase in fluorescence after addition of MTX in panel A to an exponentially rising function with rate constant k_rec. (C) Variation of the measured rate constant k_Q with the concentration of added vesicles (indicated as the concentration of externally exposed MTX-PE). (Inset) Rate constant k_rec measured at varying concentrations of added vesicles. (D) Variation of k_a (closed circles) and k_d (open circles) with the density of MBPCys bound to POPC/MTX-PE/RhoPE vesicles. Values of k_a and k_d were determined as illustrated in panel C, and in each case are scaled to the values of these parameters determined for protein-free vesicles in the same experiment. Other experimental details were as described in the text.