Cytoskeletal protein binding kinetics at planar phospholipid membranes.

Abstract

Non-specific reversible binding of cytoskeletal proteins to lipids may help guide those proteins to integral membrane anchors to the plasma membrane, or may serve as a reservoir of cytoskeletal protein immediately available for structural rearrangements at the submembrane surface. In a simplified model of reversible protein binding at cytoplasmic surfaces of living cell membranes, we studied desorption rates of the erythrocyte cytoskeletal proteins, spectrin and protein 4.1, labeled with carboxyfluoresein (CF). The model membranes consisted of planar phospholipid bilayers of phosphatidylcholine (PC) or mixed PC and phosphatidylserine (PS), supported on glass. The desorption rates were measured using the Total Internal Reflection/Fluorescence Recovery After Photobleaching (TIR/FRAP) technique. Average desorption rates for each protein were computed from multiexponential fits of the TIR/FRAP results. Equilibrium binding affinities were also obtained from TIR fluorescence photobleaching depths. Adsorption rates were calculated from the experimentally measured desorption rates and affinities. CF-spectrin has desorption rates of 3.7 s$\sp{-1}$ and 3.5 s$\sp{-1}$ for PS/PC and PC membranes, respectively, showing no preference to bind PS. CF-4.1 desorption rates differ by a factor of about two hundred: 0.008 s$\sp{-1}$ and 1.7 s$\sp{-1}$ for PS/PC and PC membranes, respectively. It is clear that protein 4.1 kinetic behavior is greatly affected by the presence of PS. Affinity alterations can result from changes in both desorption and adsorption rates. CF-spectrin binding to PS/PC membrane is enhanced by a factor of two in the presence of 4.1, and its desorption rate drops by a factor of 100 to 0.038 s$\sp{-1}$. The presence of protein 4.1 also reduced spectrin adsorption rate to PS/PC membrane. Protein 4.1 had no effect on CF-spectrin binding kinetics to the PC membrane. The presence of spectrin with CF-4.1 reduces protein 4.1 adsorption rate but has no effect on the desorption rates for both PS/PC and PC membranes. Based on these model membrane results, we speculate on the possible physiological significance, particularly on whether nonspecific binding of cytoskeletal proteins to lipids might be important in living cells and whether it might affect reaction rates in the submembrane.