Bis(sulfo-n-succinimidyl) [15N,2H16]doxyl-2-spiro-4'-pimelate, a stable isotope-substituted, membrane-impermeant bifunctional spin label for studies of the dynamics of membrane proteins: application to the anion-exchange channel in intact human erythrocytes

Abstract

We have synthesized and characterized an isotopically substituted homologue of the membrane-impermeant bifunctional spin label bis(sulfo-N-succinimidyl) doxyl-2-spiro-4'-pimelate (BSSDP) [Beth et al. (1986) Biochemistry 25, 3824-3832] in which the nitroxide N is substituted with 15N and all of the protons in the doxylpimelate moiety are replaced by deuterons ([15N,2H16]BSSDP). Like its normal isotope homologue, [15N,2H16]BSSDP reacts with the anion-exchange channel in intact human erythrocytes at a site that spans the single extracytoplasmic chymotryptic cleavage site and that overlaps the stilbenedisulfonate site. The narrower line widths in the EPR spectrum of [15N,2H16]BSDP-labeled anion channels allow calculation of a minimum separation of 16 A between spin labels bound at the functionally important stilbenedisulfonate sites on adjacent subunits of an anion channel dimer. The 15N and 2H isotopic substitutions also provide substantial improvement in signal to noise of motionally sensitive regions of the ST-EPR spectrum of [15N,2H16]BSSDP-labeled anion channels in intact erythrocytes. [15N,2H16]BSSDP-labeled anion channels in intact erythrocytes were cross-linked to covalent dimers in the extracytoplasmic domain with the membrane-impermeant cross-linking reagent bis(sulfo-N-succinimidyl) suberate [Staros (1982) Biochemistry 21, 3950-3955], and the saturation-transfer EPR spectrum of these cells was compared with that of cells treated with [15N,2H16]BSSDP but not subsequently cross-linked. The spectra were essentially identical, supporting the hypothesis that anion channel subunits form stable dimers in the membranes of intact erythrocytes.

Structural and binding properties of the stilbenedisulfonate sites on erythrocyte band 3: An electron paramagnetic resonance study using spin-labeled stilbenedisulfonates

“…Thus, EM data support the hypothesis that band 3 is dimeric, tetrameric, or some combination of the two in the membrane [reviewed in Jennings (1984)]. The dimeric association between subunits is very stable in erythrocyte membranes (Nigg & Cherry, 1979, Anjaneyulu et al, 1988, and in detergent solutions (Casey & Reithmeier, 1991). Despite the uncertainty in the oligomeric state of band 3 in situ (i.e., dimer versus tetramer), there is reasonable agreement that the dimer of band 3 is the fundamental species (Staros & Kakkad, 1983), and that this species is capable of binding stilbenedisulfonates with high affinity (Boodhoo & Reithmeier, 1984).…”

“…Convincing evidence exists that dimeric band 3 is a stable complex in intact erythrocytes (Staros, 1982;Anjaneyulu et al, 1988;Nigg & Cherry, 1979;Jennings & Nicknish, 1985).…”

“…Dipolar interactions are highly dependent on the distance and angles between neighboring nitroxide moieties. Anjaneyulu et al (1988) employed the spin-labeled crosslinker bis(sulfo-TV-succinimidyl) doxyl-2-spiro-4'-pimelate (BSSDP), an affinity label for the exofacial domain of band 3, to demonstrate a > 16-A separation between probe reaction sites on adjacent monomers.…”

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

“…Hyde and Dalton (1972), McConnell (1974), andThomas et al (1976)] demonstrated that ST-EPR spectroscopy, employing nitroxide spin-labels, provides high sensitivity to rotational motions in the micro-to millisecond time window which is the range expected for the global rotational diffusion of intrinsic membrane proteins. Numerous ST-EPR studies of membrane protein rotational dynamics have been reported [for reviews, see Thomas (1985Thomas ( , 1986], including spinlabeled band 3 (Sakaki et al, 1982;Willingham & Gaffney, 1983;Beth et al, 1986;Anjaneyulu et al, 1988;.…”

“…Saturation transfer electron paramagnetic resonance (ST-EPR) offers an alternative experimental method for the study of the rotational dynamics of membrane proteins (Thomas, 1985(Thomas, , 1986Beth & Robinson, 1989). Recently, nitroxide spin-labels which affinity label band 3 in intact erythrocytes have been reported and used to study the rotational diffusion of band 3 using ST-EPR (Beth et al, 1986;Anjaneyulu et al, 1988Anjaneyulu et al, , 1989. Most recently, an isotopically substituted spin-labeled derivative of dihydrostilbenedisulfonate ([ 15 N, 2 H 13 ]-SL-H 2 DADS-maleimide) has been reported (Wojcicki & Beth, 1993).…”