CYTOSKELETAL PROTEIN-BINDING KINETICS AT PLANAR PHOSPHOLIPID-MEMBRANES

It has been hypothesized that nonspecific reversible binding of cytosk eletal proteins to lipids in cells may guide their binding to integral membrane anchor proteins. In a model system, we measured desorption r ates k(off) (off-rates) of the erythrocyte cytoskeletal proteins spect rin and protein 4.1 labeled with carboxyfluorescein (CF), at two diffe rent compositions of planar phospholipid membranes (supported on glass ), using the total internal reflection/fluorescence recovery after pho tobleaching (TIR/FRAP) technique. The lipid membranes consisted of eit her pure phosphatidylcholine (PC) or a 3:1 mixture of PC with phosphat idylserine (PS). In general, the off-rates were not single exponential s and were fit to a combination of fast, slow, and irreversible fracti ons, reported both separately and as a weighted average, By a variatio n of TIR/FRAP, we also measured equilibrium affinities (the ratio of s urface-bound to bulk protein concentration) and thereby calculated on- rates, k(on) The average off-rate of CF-4.1 from PC/PS (similar to 0,0 08/s) is much slower than that from pure PC (similar to 1.7/s), Despit e the consequent increase in equilibrium affinity at PC/PS, the on-rat e at PC/PS is also substantially decreased (by a factor of 40) relativ e to that at pure PC. The simultaneous presence of (unlabeled) spectri n tends to substantially decrease the on-rate (and the affinity) of CF -4.1 at both membrane types, Similar experiments for CF-spectrin alone showed much less sensitivity to membrane type and generally faster of f-rates than those exhibited by CF-4,1, However, when mixed with (unla beled) 4,1, both the on-rate and off-rate of CF-spectrin decreased dra stically at PC/PS (but not PC), leading to a somewhat increased affini ty, Clearly, changes in affinity often involve countervailing changes in both on-rates and off-rates, In many of these studies, the effect o f varying ionic strength and bulk concentrations was examined; it appe ars that the binding is an electrostatic attraction and is far from sa turation at the concentrations employed, These results and the techniq ues implemented carry general implications for understanding the funct ional role of nonspecific protein binding to cellular lipid membranes.