Anisotropy decay measurement of segmental dynamics of the anion binding domain in erythrocyte band 3

Biochemistry
S BickneseA S Verkman

Abstract

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 + (ro-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 o...Continue Reading

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Citations

Jun 19, 2003·Photochemistry and Photobiology·Nectarios Klonis, William H Sawyer
Oct 14, 2004·Molecular and Biochemical Parasitology·Enrique WinogradIrwin W Sherman

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