Determination of the Orientation of a Band 3 Affinity Spin-Label Relative to the Membrane Normal Axis of the Human Erythrocyte

Abstract

The orientation of the nitroxide moiety of an isotopically substituted spin-labeled derivative of dihydrostilbenedisulfonate ([15N,2H13]-SL-H2DADS-maleimide) covalently coupled at the extracellular stilbenedisulfonate binding site of the human erythrocyte anion exchange protein, band 3, has been determined relative to the membrane normal axis of intact cells. The X-band linear electron paramagnetic resonance (EPR) spectra of [15N,2H13]-SL-H2DADS-maleimide-labeled band 3 in intact erythrocytes oriented by flow through an EPR flat cell have been obtained for two orthogonal orientations of the sample in the DC magnetic field. Two different methods of analysis have provided very similar values for the angles alpha 1 and beta 1 which uniquely define the orientation of the nitroxide axis frame relative to the membrane normal axis. In the first approach, a variable fraction of the cells, f, were taken to be biconcave disks perfectly oriented relative to the flat cell surface with the remainder, 1-f, isotropically oriented. Simultaneous nonlinear least squares analysis of the spectra obtained at the two sample orientations yielded best fit values of f = 0.60, alpha 1 = 58 degrees, and beta 1 = 36 degrees. In the second approach, the EPR spectra of flow-oriented intact erythrocytes labeled with the fatty acid spin-label, [15N,2H12]-5-nitroxyl stearate, have been obtained at the two sample orientations. These two spectra have been used to determine a model-independent distribution of membrane normal orientations in the sample. Using this experimentally determined membrane normal orientation distribution, the EPR spectra of [15N,2H13]-SL-H2DADS-maleimide-labeled erythrocytes were then reanalyzed to obtain a second determination of the nitroxide orientation, alpha 1 = 61 degrees and beta 1 = 37 degrees. The orientation of the nitroxide with respect to the membrane normal axis determined in the present study is nearly identical to the orientation of the nitroxide with respect to the uniaxial rotational diffusion axis, alpha = 66 degrees and beta = 34 degrees, as determined from saturation transfer EPR (ST-EPR) studies [Hustedt, E. H., & Beth, A. H. (1995) Biophys. J. 69, 1409-1423]. This result supports the conclusion that the motion observed using ST-EPR spectroscopy is, in fact, the uniaxial rotational diffusion of band 3 about the membrane normal.

The Sensitivity of Saturation Transfer Electron Paramagnetic Resonance Spectra to Restricted Amplitude Uniaxial Rotational Diffusion

“…By incorporating this algorithm into a nonlinear least-squares routine based upon the Marquardt-Levenberg approach, the rotational correlation time for spin-labeled band 3 in erythrocyte membrane preparations was determined by optimizing the fit between experimental and calculated spectra (Hustedt and Beth, 1995). Although this work provided evidence that the vast majority of copies of band 3 were undergoing URD about the membrane normal axis (Hustedt and Beth, 1996) of reasonably large amplitude and a rate that was consistent with dimers or tetramers, it remained uncertain whether a constraint to rotational motion might be overlooked in this simple analysis. This concern was based upon the known interactions between the cytoplasmic domain of a subpopulation of band 3 and the membrane skeleton from a wide range of studies (see Low, 1986) and from previous TOA studies that demonstrated a large change in anisotropy decay of the transmembrane domain of band 3 following proteolytic cleavage of the link with the cytoplasmic domain (Nigg and Cherry, 1980).…”

The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization microscopy

“…Recent EPR studies by Hustedt and Beth (1996) have indicated that the major rotational diffusion axis of band 3, as reported by a spin-labeled stilbenedisulfonate, is coincident with the membrane normal axis. Therefore, as a starting point for a more detailed investigation of the multiexponential anisotropy decays of band 3, a valid assumption would be that the orientation of eosin relative to the diffusion axis is equivalent to its orientation relative to the membrane normal axis.…”

Flexibility of the Cytoplasmic Domain of the Anion Exchange Protein, Band 3, in Human Erythrocytes

“…To accomplish this goal, experimental data from labeled band 3 have been obtained and analyzed in terms of an internally consistent model using both TOA and ST-EPR. The computational algorithm described in Hustedt and Beth (2001) has been employed to define the spectral sensitivity of ST-EPR at X-and Q-band microwave frequencies to the amplitude of URD using the spin-label orientation that has been previously determined for spin-labeled band 3 (Hustedt and Beth, 1996). Calculations indicate that spectral sensitivity is greatly diminished even for a single homogeneous population of band 3 as the amplitude of URD exceeds ϳ60°at both microwave frequencies.…”