Time-resolved anisotropy was utilized to detect nanosecond segmental motions of the band 3 intramembrane domain. Band 3 at lysine 430 was fluorescently labeled in ghost membranes by fluorescein or eosin maleimide treatment of intact human erythrocytes followed by hypotonic lysis. Single lifetimes for fluorescein (3.8-4.1 ns) and eosin (3.2-3.4 ns) were observed. Phase-modulation measurement of anisotropy decay indicated a segmental motion model, r(t) = exp(-t/tau(1c))[r(infinity) + (r(o) - r(infinity)) exp(-t/tau(2c))], defined by rotational correlation times corresponding to band 3 segmental motion (tau(1c), 30-70 ns) and rapid fluorescein motion in its binding pocket (tau(2c), 200-400 ps), and a residual anisotropy (r(infinity), 0.23-0.28) describing hindered fluorescein motion. In PBS at pH 7.4, tau(1c), tau(2c), and r(infinity) were 44 ns, 307 ps, and 0.24, respectively, predicting a steady-state anisotropy of 0.24, in agreement with the measured value of 0.23. Factors that might influence band 3 structure/dynamics were examined. Whereas pH (range 5-10) had little effect on r(t), [NaCl] addition (0-150 mM) remarkably decreased tau(1c) from 68 to 44 ns. The decrease in tau(1c) correlated with solution ionic strength, and did not depend on osmolality (studied by mannitol addition), or specific anion interactions (comparing Cl, Br, F, SO4, citrate). The ionic strength effect was not observed in fluorescein-labeled carbonic anhydrase and trypsin-cleaved band 3, suggesting a specific effect on intact band 3. Anisotropy decay was relatively insensitive to external lectin or internal 2,3-DPG binding, but was sensitive to temperature, membrane fluidity, urea denaturation, fluid-phase viscosity, and aldehyde fixation. These studies provide the first data on the nanosecond segmental dynamics of the intramembrane domain of band 3 and revealed an unexpected sensitivity to solution ionic strength.
