title: Flexibility of the cytoplasmic domain of the anion exchange protein, band 3, in human erythrocytes.
abstract: The rotational flexibility of the cytoplasmic domain of band 3, in the region that is proximal to the inner membrane surface, has been investigated using a combination of time-resolved optical anisotropy (TOA) and saturation-transfer electron paramagnetic resonance (ST-EPR) spectroscopies. TOA studies of rotational diffusion of the transmembrane domain of band 3 show a dramatic decrease in residual anisotropy following cleavage of the link with the cytoplasmic domain by trypsin (E. A. Nigg and R. J. Cherry, 1980, Proc. Natl. Acad. Sci. U.S.A. 77:4702-4706). This result is compatible with two independent hypotheses: 1) trypsin cleavage leads to dissociation of large clusters of band 3 that are immobile on the millisecond time scale, or 2) trypsin cleavage leads to release of a constraint to uniaxial rotational diffusion of the transmembrane domain. ST-EPR studies at X- and Q-band microwave frequencies detect rotational diffusion of the transmembrane domain of band 3 about the membrane normal axis of reasonably large amplitude that does not change upon cleavage with trypsin. These ST-EPR results are not consistent with dissociation of clusters of band 3 as a result of cleavage with trypsin. Global analyses of the ST-EPR data using a newly developed algorithm indicate that any constraint to rotational diffusion of the transmembrane domain of band 3 via interactions of the cytoplasmic domain with the membrane skeleton must be sufficiently weak to allow rotational excursions in excess of 32 degrees full-width for a square-well potential. In support of this result, analyses of the TOA data in terms of restricted amplitude uniaxial rotational diffusion models suggest that the membrane-spanning domain of that population of band 3 that is linked to the membrane skeleton is constrained to diffuse in a square-well of approximately 73 degrees full-width. This degree of flexibility may be necessary for providing the unique mechanical properties of the erythrocyte membrane.
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors.forename: S M
authors.initials: SM
authors.lastname: Blackman
authors.forename: E J
authors.initials: EJ
authors.lastname: Hustedt
authors.forename: C E
authors.initials: CE
authors.lastname: Cobb
authors.forename: A H
authors.initials: AH
authors.lastname: Beth
mesh_terms: D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry*; D016880:Anisotropy; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry* / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D008956:Models, Chemical; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D014357:Trypsin