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.

Sulfate influx on band 3 protein of equine erythrocyte membrane (Equus caballus) using different experimental temperatures and buffer solutions

“…The results presented in this article showed the functional aspects of the anion channel protein in horse. As previously observed in other species, 29,15,30 the structural integrity of band 3 has a close relationship with the anion transport in horse too. The different experimental conditions, in fact, reflect in alterations, which concern the structure of the membrane in all its components and this also influences sulfate influx.…”

Imaging of the diffusion of single band 3 molecules on normal and mutant erythrocytes

“…Rotational relaxation studies of Cherry and colleagues 45 demonstrated that both populations were mobile, but that one exhibited relatively unrestricted rotation and the other displayed restrained, yet measurable rotation. Subsequent studies by Beth and coworkers 46 showed that the skeletally attached band 3 can rotate through an angle of 73°, probably because of the flexibility of the junction between the cytoplasmic and membrane-spanning domains of band 3. Data of Golan et al 47 have further demonstrated that depletion of ankyrin releases the less mobile population from its rotational restriction and that the rotational and lateral diffusion of band 3 are independently regulated, the former by protein interactions at the cytoplasmic surface of the membrane, and the latter by the spectrin content of the cell.…”

Changes in Band 3 oligomeric state precede cell membrane phospholipid loss during blood bank storage of red blood cells

“…We also labeled Band 3 on the RBC membrane with eosin‐5‐maleimide (EMA) to perform time‐resolved phosphorescence anisotropy (TPA) and fluorescence resonance energy transfer (FRET) experiments on the same 5 RBC units on Days 0, 12, 20, 29, 42, and 49 of cold storage. TPA has previously been shown to resolve distinct populations of Band 3 oligomeric complexes in the RBC membrane and is thus a powerful spectroscopic tool to determine the effect of RBC storage on the organization of membrane proteins 13,14 . Fluorescence resonance energy homotransfer is an independent (compared to TPA) technique that also detects large changes in the organization of Band 3 in the RBC membrane 15 .…”