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

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.     

Bis(sulfo-N-succinimidyl) doxyl-2-spiro-5'-azelate: synthesis, characterization, and reaction with the anion-exchange channel in intact human erythrocytes

We have synthesized and characterized bis(sulfo-N-succinimidyl) doxyl-2-spiro-5'-azelate (BSSDA), a membrane-impermeant bifunctional spin-labeling reagent. BSSDA is a nine carbon backbone homologue of bis(sulfo-N-succinimidyl) doxyl-2-spiro-4'-pimelate [BSSDP; Beth et al. (1986) Biochemistry 25, 3824-3832]. Due to its longer backbone, BSSDA can span longer distances between reactive groups on a protein than can BSSDP. However, the purpose of the bifunctional design of these reagents is to provide a tight motional coupling of the spin labels to the surface of a target protein. To test whether the longer backbone of BSSDA results in a greater local flexibility and thereby undermines the effects of bidentate attachment, we have labeled with BSSDA anion-exchange channels of intact human erythrocytes at the same site as we have previously labeled them with BSSDP. Linear and saturation-transfer EPR spectra of BSSDA-labeled anion-exchange channels in intact cells closely approximate the corresponding spectra from BSSDP-labeled channels. Thus, the longer backbone of BSSDA relative to BSSDP does not give rise to significant local flexibility, even when BSSDA is bound to a site that can be spanned by the shorter reagent.     

Effects of diethyl ether on membrane lipid ordering and on rotational dynamics of the anion exchange protein in intact human erythrocytes: correlations with anion exchange function

The roles of lipid ordering and protein dynamics on the function of the anion exchange protein (band 3) in intact human erythrocytes have been investigated. The effects of diethyl ether on the ordering of membrane lipids and on the rotational dynamics of band 3 were measured by EPR and saturation-transfer EPR spectroscopies, respectively, and correlated with the anion exchange function of band 3. With increasing concentration, diethyl ether monotonically decreased the ordering of membrane lipids near the polar head-group region, as reported by the lipid-soluble spin probe 5-doxylstearic acid, but produced comparatively little change in the ordering of lipids in the hydrophobic midzone, as reported by 16-doxylstearic acid. The rotational mobility of band 3, as reported by the affinity spin-label bis(sulfo-N-succinimidyl) doxyl-2-spiro-5'-azelate [Anjaneyulu et al. (1989) Biochemistry 28, 6583-6590], also increased monotonically with increasing ether concentration. This increase in rotational mobility was not due to a demonstrable change in its state of oligomerization, since band 3 was readily cross-linked by bis(sulfo-N-succinimidyl) suberate to covalent dimers in the presence or absence of ether. At concentrations up to 2 vol % ether, hemolysis of erythrocytes was negligible, and the spectroscopic changes observed were completely reversed following its removal. Km, Vmax, and Eact. for sulfate uptake into chloride-loaded erythrocytes were not significantly affected by addition of ether.(ABSTRACT TRUNCATED AT 250 WORDS)     

Saturation-transfer electron paramagnetic resonance detection of anisotropic motion by sickle hemoglobin molecules in the polymer state

The motional behavior of spin-labeled deoxygenated sickle hemoglobin has been studied by using both 9- and 35-GHz saturation-transfer electron paramagnetic resonance (EPR). Using spectral subtraction techniques and saturation-transfer EPR parameter correlation plots, we find that the saturation-transfer EPR spectra for the sickle hemoglobin gel state at high temperature and high hemoglobin concentration cannot be described as a simple superposition of spectra from immobilized hemoglobin plus solution-state hemoglobin but instead suggest that the individual sickle hemoglobin molecules exhibit limited, anisotropic, rotational oscillation within the polymer fiber. The spectra also imply that the symmetry axis for sickle hemoglobin rotational oscillation is approximately coincident with the nitroxide z axis of the covalently attached spin-label. We suggest that this anisotropic rotational motion may be produced by one or two of the known intermolecular contact sites within the sickle hemoglobin fiber acting as strong intermolecular binding sites, and producing "motional alignment" within the fiber; determining the location of the strong binding site should be important in focusing the future development of antisickling agents.     

Cross-linking and chymotryptic digestion of the extractoplasmic domain of the anion exchange channel in intact human erythrocytes

We have applied our new high yield, membrane-impermeant, protein cross-linking reagents (J.V. Staros, 1982. Biochemistry 21:3950-3955) together with chymotryptic digestion of the surface of intact erythrocytes (T.L. Steck, B. Ramos, and E. Strapazon, 1976. Biochemistry 15:1154-1161) in an investigation of the topology of the extracytoplasmic domain of the anion exchange channel of intact human erythrocytes. In intact erythrocytes, these cross-linking reagents have been shown to cross-link subunits of the anion exchange channel to dimers in the extracytoplasmic domain of the protein. Chymotryptic treatment of intact erythrocytes has been shown to cleave subunits of the anion exchange channel into two fragments of distinct Mr. Sequential treatment of intact erythrocytes with either of two membrane-impermeant cross-linkers, followed by digestion with chymotrypsin, yields chymotryptic fragments of the anion exchange channel cross-linked to one another. The cross-linked products observed appear to arise by cross-linking of unlike chymotryptic fragments, whether the cross-links are intersubunit or intrasubunit. These results are consistent with a model of the anion exchange channel in which the subunits form a head-to-head dimer with a twofold center of symmetry perpendicular to the plane of the membrane.     

Saturation transfer electron paramagnetic resonance study of the mobility of myosin heads in myofibrils under conditions of partial dissociation.

The rotational motion of rigidly spin-labeled myosin heads of glycerinated myofibrils as reflected in saturation-transfer EPR spectra behaves to a first approximation as though the heads consist of two populations with different rotational motions. An immobilized fraction has a correlation time (tau 2) of approximately 0.5 ms, comparable to that of spin-labeled subfragment-1 (S1) bound to thin filaments, while a mobile fraction has a tau 2 of 10 microseconds, comparable to that of the heads of purified myosin filaments. The effects of nonhydrolyzable ATP analogues, potassium pyrophosphate (PPi), or adenylyl imidodiphosphate, Ca2+, temperature, or ionic strength on the spectra can be analyzed in terms of the fraction of myosin heads immobilized by attachment to thin filaments, without requiring changes in the motion of either attached or detached heads.     

Identification of the eosinyl-5-maleimide reaction site on the human erythrocyte anion-exchange protein: overlap with the reaction sites of other chemical probes.

The anion-exchange protein (band 3) reaction site in human erythrocytes for the fluorescent/phosphorescent probe eosinyl-5-maleimide (EMA) has been identified. Proteolytic dissection of band 3 in situ indicated that EMA reacts with the membrane-spanning Mr 17K peptide produced by chymotrypsin cleavage of band 3 in intact erythrocytes followed by removal of the cytoplasmic domain by mild trypsin digestion of ghost membranes. Sequencing of the major eosin-labeled peptide obtained from HPLC purification of an extensive chymotrypsin digest of purified Mr 17K peptide allowed assignment of the covalent reaction site for EMA to lysine-430 of the human erythrocyte protein [Tanner et al. (1988) Biochem. J. 256, 703-712]. Hydropathy plots based upon the primary structure of the protein [Lux et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9089-9093] suggest that this residue is in an extracellularly accessible loop connecting membrane-spanning segments 1 and 2 of native band 3 in the erythrocyte membrane. Inhibition of sequential labeling of intact erythrocytes by pairs of chemical probes including EMA, the anion transport inhibitor 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate (H2-DIDS), and the reactively bifunctional spin-label bis(sulfo-N-succinimidyl) doxyl-2-spiro-5'-azelate (BSSDA) has also been investigated. Each of these reagents affinity labels band 3 when added separately to a suspension of intact human erythrocytes by formation of one or more stable covalent bonds. Prelabeling of intact erythrocytes with EMA reduced subsequent labeling of band 3 by H2-DIDS by approximately 95% and by BSSDA by 90%. Similarly, prelabeling with H2-DIDS reduced subsequent labeling of band 3 by EMA by over 90%, and BSSDA prelabeling reduced EMA labeling by approximately 95%. Therefore, though having widely divergent chemical structures and protein modification reactivities, each of these negatively charged reagents may be competing for reaction with spatially overlapping sites on band 3 which are accessible from the extracellular space.     

Temperature dependence of rotational dynamics of protein and lipid in sarcoplasmic reticulum membranes

We have investigated the relationship between function and molecular dynamics of both the lipid and the Ca-ATPase protein in sarcoplasmic reticulum (SR), using temperature as a means of altering both activity and rotational dynamics. Conventional and saturation-transfer electron paramagnetic resonance (EPR) was used to probe rotational motions of spin-labels attached either to fatty acid hydrocarbon chains or to the Ca-ATPase sulfhydryl groups in SR. EPR studies were also performed on aqueous dispersions of extracted SR lipids, in order to study intrinsic lipid properties independent of the protein. While an Arrhenius plot of the Ca-ATPase activity exhibits a clear change in slope at 20 degrees C, Arrhenius plots of lipid hydrocarbon chain mobility are linear, indicating that an abrupt thermotropic change in the lipid hydrocarbon phase is not responsible for the Arrhenius break in enzymatic activity. The presence of protein was found to decrease the average hydrocarbon chain mobility, but linear Arrhenius plots were observed both in the intact SR and in extracted lipids. Lipid EPR spectra were analyzed by procedures that prevent the production of artifactual breaks in the Arrhenius plots. Similarly, using sample preparations and spectral analysis methods that minimize the temperature-dependent contribution of local probe mobility to the spectra of spin-labeled Ca-ATPase, we find that Arrhenius plots of overall protein rotational mobility also exhibit no change in slope. The activation energy for protein mobility is the same as that of ATPase activity above 20 degrees C; we discuss the possibility that overall protein mobility may be essential to the rate-limiting step above 20 degrees C.     

Rotational diffusion of the erythrocyte integral membrane protein band 3: effect of hemichrome binding.

Human erythrocyte band 3 was covalently labeled within the integral membrane domain by incubating intact erythrocytes with the phosphorescent probe eosinyl-5-maleimide. The rotational diffusion of band 3 in membranes prepared from these labeled cells was measured using the technique of time-resolved phosphorescence anisotropy. Three rotational correlation times ranging from 16 to 3800 microseconds were observed, suggesting that band 3 exists in different aggregate states within the plane of the membrane. The oxidizing agent phenylhydrazine was used to induce hemichrome formation within intact erythrocytes. The immobilization of band 3 in membranes prepared from these erythrocytes suggests that the binding of hemichromes induces clustering of band 3. The addition of purified hemichromes to erythrocyte ghosts leads to a similar effect. We have also examined the mobility of the cytoplasmic domain of band 3. This region was labeled indirectly using a phosphorescently labeled antibody which binds to an epitope within the cytoplasmic domain. We observed very rapid motion of the cytoplasmic region of band 3, which was only partially restricted upon hemichrome binding. This suggests that the integral and cytoplasmic domains of band 3 may be independently mobile.     

Effects of ouabain on the rotational dynamics of renal Na,K-ATPase studied by saturation-transfer EPR.

The interaction of a nitroxide spin-labeled derivative of ouabain with sheep kidney Na,K-ATPase and the motional behavior of the ouabain spin label-Na,K-ATPase complex have been studied by means of electron paramagnetic resonance (EPR) and saturation-transfer EPR (ST-EPR). Spin-labeled ouabain binds with high affinity to the Na,K-ATPase with concurrent inhibition of ATPase activity. Enzyme preparations retain 0.61 +/- 0.1 mol of bound ouabain spin label per mole of ATP-dependent phosphorylation sites, even after repeated centrifugation and resuspension of the purified ATPase-containing membrane fragments. The conventional EPR spectrum of the ouabain spin label bound to the ATPase consists almost entirely (greater than 99%) of a broad resonance at 0 degrees C, characteristic of a tightly bound spin label which is strongly immobilized by the protein backbone. Saturation-transfer EPR measurements of the spin-labeled ATPase preparations yield effective correlation times for the bound labels significantly longer than 100 microseconds at 0 degrees C. Since the conventional EPR measurements of the ouabain spin-labeled Na,K-ATPase indicated the label was strongly immobilized, these rotational correlation times most likely represent the motion of the protein itself rather than the independent motion of mobile spin probes relative to a slower moving protein. Additional ST-EPR measurements of ouabain spin-labeled Na,K-ATPase (a) cross-linked with glutaraldehyde and (b) crystallized in two-dimensional arrays indicated that the observed rotational correlation times predominantly represented the motion of large Na,K-ATPase-containing membrane fragments, as opposed to the motion of individual monomeric or dimeric polypeptides within the membrane fragment. The results suggest that the binding of spin-labeled ouabain to the ATPase induces the protein to form large aggregates, implying that cardiac glycoside induced enzyme aggregation may play a role in the mechanism of action of the cardiac glycosides in inhibiting the Na,K-ATPase.     

Location of the stilbenedisulfonate binding site of the human erythrocyte anion-exchange system by resonance energy transfer.

The stilbenedisulfonate inhibitory site of the human erythrocyte anion-exchange system has been characterized by using serveral fluorescent stilbenedisulfonates. The covalent inhibitor 4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate (BIDS) reacts specifically with the band 3 protein of the plasma membrane when added to intact erythrocytes, and the reversible inhibitors 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) and 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS) show a fluorescence enhancement upon binding to the inhibitory site on erythrocyte ghosts. The fluorescence properties of all three bound probes indicate a rigid, hydrophobic site with nearby tryptophan residues. The Triton X-100 solublized and purified band 3 protein has similar affinities for DBDS, BADS, and 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) to those observed on intact erythrocytes and erythrocyte ghosts, showing that the anion binding site is not perturbed by the solubilization procedure. The distance between the stilbenedisulfonate binding site and a group of cysteine residues on the 40 000-dalton amino-terminal cytoplasmic domain of band 3 was measured by the fluorescence resonance energy transfer technique. Four different fluorescent sulfhydryl reagents were used as either energy transfer donors or energy transfer acceptors in combination with the stilbenedisulfonates (BIDS, DBDS, BADS, and DNDS). Efficiencies of transfer were measured by sensitized emisssion, donor quenching, and donor lifetime changes. Although these sites are approachable from opposite sides of the membrane by impermeant reagents, they are separated by only 34--42 A, indicating that the anion binding site is located in a protein cleft which extends some distance into the membrane.     

Flexibility of the cytoplasmic domain of the anion exchange protein, band 3, in human erythrocytes.

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.     

Analysis of saturation transfer electron paramagnetic resonance spectra of a spin-labeled integral membrane protein, band 3, in terms of the uniaxial rotational diffusion model.

Algorithms have been developed for the calculation of saturation transfer electron paramagnetic resonance (ST-EPR) spectra of a nitroxide spin-label assuming uniaxial rotational diffusion, a model that is frequently used to describe the global rotational dynamics of large integral membrane proteins. One algorithm explicitly includes terms describing Zeeman overmodulation effects, whereas the second more rapid algorithm treats these effects approximately using modified electron spin-lattice and spin-spin relaxation times. Simulations are presented to demonstrate the sensitivity of X-band ST-EPR spectra to the rate of uniaxial rotational diffusion and the orientation of the nitroxide probe with respect to the diffusion axis. Results obtained by using the algorithms presented, which are based on the transition-rate formalism, are in close agreement with those obtained by using an eigenfunction expansion approach. The effects of various approximations used in the simulation algorithms are considered in detail. Optimizing the transition-rate formalism to model uniaxial rotational diffusion results in over an order of magnitude reduction in computation time while allowing treatment of nonaxial A- and g-tensors. The algorithms presented here are used to perform nonlinear least-squares analyses of ST-EPR spectra of the anion exchange protein of the human erythrocyte membrane, band 3, which has been affinity spin-labeled with a recently developed dihydrostilbene disulfonate derivative, [15N,2H13]-SL-H2DADS-MAL. These results suggest that all copies of band 3 present in intact erythrocytes undergo rotational diffusion about the membrane normal axis at a rate consistent with a band 3 dimer.     

Saturation transfer electron parametric resonance of an indane-dione spin-label. Calibration with hemoglobin and application to myosin rotational dynamics.

We have used a recently synthesized indane-dione spin label (2-[-oxyl-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methenyl]in dane-1,3-dione (InVSL) to study the rotational dynamics of myosin, with saturation-transfer electron paramagnetic resonance (ST-EPR). To determine effective rotational correlation times (tau effr) from InVSL spectra, reference spectra corresponding to known correlation times (tau r) were obtained from InVSL-hemoglobin undergoing isotropic rotational motion in aqueous glycerol solutions. These spectra were used to generate plots of spectral parameters vs. tau r. These plots should be used to analyze ST-EPR spectra of InVSL bound to other proteins, because the spectra are different from those of tempo-maleimide-spin-labeled hemoglobin, which have been used previously as ST-EPR standards. InVSL was covalently attached to the head (subfragment-1; S1) of myosin. EPR spectra and K/EDTA-ATPase activity showed that 70-95% of the heads were labeled, with > or = 90% of the label bound to either cys 707 (SH1) or cys 697 (SH2). ST-EPR spectra of InVSL-S1 attached to glass beads, bound to actin in myofibrils, or precipitated with ammonium sulfate indicated no submillisecond rotational motion. Therefore, InVSL is rigidly immobilized on the protein so that it reports the global rotation of the myosin head. The ST-EPR spectra of InVSL-myosin monomers and filaments indicated tau effr values of 4 and 13 microseconds, respectively, showing that myosin heads undergo microsecond segmental rotations that are more restricted in filaments than in monomers. The observed tau effr values are longer than those previously obtained with other spin labels bound to myosin heads, probably because InVSL binds more rigidly to the protein and/or with a different orientation. Further EPR studies of InVSL-myosin in solution and in muscle fibers should prove complementary to previous work with other labels.     

Erythrocyte signal transduction pathways, their oxygenation dependence and functional significance.

Erythrocytes play a key role in human and vertebrate metabolism. Tissue O2 supply is regulated by both hemoglobin (Hb)-O2 affinity and erythrocyte rheology, a key determinant of tissue perfusion. Oxygenation-deoxygenation transitions of Hb may lead to re-organization of the cytoskeleton and signalling pathways activation/deactivation in an O2-dependent manner. Deoxygenated Hb binds to the cytoplasmic domain of the anion exchanger band 3, which is anchored to the cytoskeleton, and is considered a major mechanism underlying the oxygenation-dependence of several erythrocyte functions. This work discusses the multiple modes of Hb-cytoskeleton interactions. In addition, it reviews the effects of Mg2+, 2,3-diphosphoglycerate, NO, shear stress and Ca2+, all factors accompanying the oxygenation-deoxygenation cycle in circulating red cells. Due to the extensive literature on the subject, the data discussed here, pertain mainly to human erythrocytes whose O2 affinity is modulated by 2,3-diphosphoglycerate, ectothermic vertebrate erythrocytes that use ATP, and to bird erythrocytes that use inositol pentaphosphate.     

A new bifunctional spin-label suitable for saturation-transfer EPR studies of protein rotational motion

A new bifunctional spin-label (BSL) has been synthesized that can be immobilized on the surface of proteins, allowing measurement of rotational motion of proteins by saturation-transfer electron paramagnetic resonance (STEPR). The spin-label contains a photoactivatable azido moiety, a cleavable disulfide, and a nitroxide spin with restricted mobility relative to the rest of the label. The label reacts with surface lysine residues modified with beta-mercaptopropionate. Bifunctional attachment is achieved by photoactivation of the azido group. Any spin-label that remains monofunctionally attached after photolysis is removed by reduction of the disulfide. Only bifunctionally attached BSL remains on the protein. Hemoglobin was used to test the utility of the BSL in STEPR by comparison with hemoglobin modified with maleimide spin-label (MSL), a commonly used standard for the STEPR technique. MSL is a monofunctional spin-label which is fortuitously immobilized by local protein structure within hemoglobin. The BSL labeling of hemoglobin did not significantly affect the quaternary structure of hemoglobin as determined by gel filtration chromatography. The conventional EPR spectra of the mono- and bifunctionally attached BSL-hemoglobin were similar to the MSL-hemoglobin spectrum, indicating that both forms of BSL were rigidly bound to hemoglobin. In contrast, the spectrum obtained by reaction of modified hemoglobin lysine residues with MSL indicated that these labels were highly mobile. The monofunctionally attached BSL was mobilized upon octyl glucoside addition whereas bifunctionally attached BSL was only slightly mobilized, suggesting that hydrophobic interactions immobilize the monofunctionally attached label on hemoglobin. The response of STEPR spectra of mono- and bifunctionally attached BSL-hemoglobin to changes in hemoglobin rotational correlation time was similar to the MSL-hemoglobin over the range of 10(-5)-10(-3) s. The spectra of bifunctionally attached BSL indicated slightly less motion than corresponding spectra for MSL or monofunctionally attached BSL. The new BSL is a good reporter of protein rotation and does not require unique protein structures for its immobilization on the protein. Thus, the BSL should be more generally applicable for STEPR studies of membrane protein rotation than existing monofunctional spin-labels.     

Interactions of actin and tubulin with human deoxyhemoglobin. Their possible occurrence within erythrocytes

Short actin filaments are an essential component of the red-cell membrane skeleton, and microtubules are also present in nucleated erythrocytes as a marginal band. Actin and tubulin share the property of possessing a very anionic terminal peptide. Since deoxyhemoglobin (Hb) is known to be a strong polyanion-binding protein, we have considered how it may interact with actin and tubulin within the intact cell. Here we demonstrate that actin and tubulin form in vitro a high-affinity complex with Hb. This is shown by measuring, by stopped-flow experiments, the decrease of the binding rate constant of CO to Hb in the presence of increasing amounts of actin and tubulin. One tetramer of Hb is bound by an actin monomer, and about two tetramers by an alpha, beta-tubulin heterodimer. Binding assays in batch experiments with immobilized tubulin give the same stoichiometry. Formation of the complexes involves the 2,3-bisphosphoglycerate-binding site of Hb and a negatively charged domain, most likely the highly acidic N and C-terminal peptides of actin and tubulin. In addition to providing new opportunities to study the structural and functional properties of actin and tubulin, these results support the idea that in the case of partial metabolic depletion of bisphosphoglycerate and ATP in erythrocytes, Hb may interact with oligomeric actin and tubulin present in the cytoskeleton.     

Strong interactions between a spin-labeled cholesterol analog and erythrocyte proteins in the human erythrocyte membrane

We have used a spin label analog of cholesterol bearing a nitroxide on the alkyl chain (26-nor-25-doxylcholestanol) to study cholesterol-protein interactions in the human erythrocyte membrane. As judged from the ESR spectrum, the spin label is readily incorporated into the membrane when added from a concentrated ethanolic solution to a cell or ghost suspension. With intact erythrocytes or white ghosts in isotonic buffer, the ESR spectrum is a superposition of a mobile component and a strongly immobilized component (outer hyperfine splitting 61–63 G). The latter corresponds to approx. 45% of the signal, a percentage which is barely affected by varying the temperature between 5 and 37°C. Removal of the cytoskeletal proteins spectrin and actin by low ionic strength treatment or of all extrinsic proteins by alkali treatment of ghosts reduces the immobilized fraction to approx. 25%. The effect of controlled proteolysis of intrinsic proteins was also tested. Pre-treatment of cells with chymotrypsin or pre-treatment of unsealed ghosts with trypsin has no effect on the ESR spectrum obtained with alkali-treated membranes. On the other hand, after chymotrypsin treatment of unsealed ghost, which reduces the band 3 protein to a 17.5 kDa membrane fragment, the strongly immobilized component is no longer observable. These data show that the cholesterol analog 26-nor-25-doxylcholestanol interacts strongly with one or several proteins of the erythrocyte membrane. That the intrinsic protein band 3 is involved is suggested by the disappearance of the immobilized fraction occurring upon chymotrypsin digestion of this protein. Our results are thus consistent with the proposal of a selective cholesterol-band 3 interaction in the erythrocyte membrane (Schubert, D. and Boss, K. (1982) FEBS Lett. 150, 4–8). Our data also suggest that this interaction is influenced by cytoskeletal proteins, an effect which can be explained considering the known linking of band 3 to the erythrocyte cytoskeleton via ankyrin. Experiments have also been carried out with 3-doxylandrostanol, a more commonly used cholesterol spin-label analog. With this spin label, at all temperatures investigated, we found it impossible to demonstrate unambiguously the existence of two separate spectral components. It is suggested that 26-nor-25-doxylcholestanol is a better reporter of cholesterol behavior in membranes.     

Lipid fluidity directly modulates the overall protein rotational mobility of the Ca-ATPase in sarcoplasmic reticulum

We have developed a quantitative and relatively model-independent measure of lipid fluidity using EPR and have applied this method to compare the temperature dependence of lipid hydrocarbon chain fluidity, overall protein rotational mobility, and the calcium-dependent enzymatic activity of the Ca-ATPase in sarcoplasmic reticulum. We define membrane lipid fluidity to be T/eta, where eta is the viscosity of a long chain hydrocarbon reference solvent in which a fatty acid spin label gives the same EPR spectrum (quantitated by the order parameter S) as observed for the same probe in the membrane. This measure is independent of the reference solvent used as long as the spectral line shapes in the membrane and the solvent match precisely, indicating that the same type of anisotropic probe motion occurs in the two systems. We argue that this empirical measurement of fluidity, defined in analogy to the macroscopic fluidity (T/eta) of a bulk solvent, should be more directly related to protein rotational mobility (and thus to protein function) than are more conventional measures of fluidity, such as the rate or amplitude of rotational motion of the lipid hydrocarbon chains themselves. This new definition thus offers a fluidity measure that is more directly relevant to the protein's behavior. The direct relationship between this measure of membrane fluidity and protein rotational mobility is supported by measurements in sarcoplasmic reticulum. The overall rotational motion of the spin-labeled Ca-ATPase protein was measured by saturation-transfer EPR. The Arrhenius activation energy for protein rotational mobility (11-12 kcal/mol/degree) agrees well with the activation energy for lipid fluidity, if defined as in this study, but not if more conventional definitions of lipid fluidity are used. This agreement, which extends over the entire temperature range from 0 to 40 degrees C, suggests that protein mobility depends directly on lipid fluidity in this system, as predicted from hydrodynamic theory. The same activation energy is observed for the calcium-dependent ATPase activity under physiological conditions, suggesting that protein rotational mobility (dependent on lipid fluidity) is involved in the rate-limiting step of active calcium transport.     

Rotational dynamics of lipid and the Ca-ATPase in sarcoplasmic reticulum. The molecular basis of activation by diethyl ether

We have investigated the role of lipid and protein dynamics in the activation of the Ca2+-dependent ATPase in sarcoplasmic reticulum (SR) by diethyl ether. Conventional and saturation-transfer electron paramagnetic resonance (EPR) were used to probe rotational motions of spin labels attached either to fatty acid hydrocarbon chains or to the Ca-ATPase in SR. We confirm previous studies (Salama, G., and Scarpa, A. (1980) J. Biol. Chem. 255, 6525-6528; Salama, G., and Scarpa, A. (1983) Biochem. Pharmacol. 32, 3465-3477; Kidd, A., Scales, D., and Inesi, G. (1981) Biochem. Biophys. Acta 65, 124-131) reporting that addition of diethyl ether to SR results in an approximately 2-fold enzymatic activation, without loss of coupling. Diethyl ether progressively fluidizes the SR membrane with respect to lipid hydrocarbon chain dynamics probed at several depths in the bilayer. Digital substractions, used to analyze two-component lipid spin label spectra, reveal that a 2-fold mobilization occurs in the population of lipid probes motionally restricted by the protein, while the remaining more mobile population is less affected. The microwave saturation properties of lipid probes also indicate that restricted motions of these probes are mobilized in maximally activated SR membranes. Saturation-transfer EPR, applied to maleimide spin-labeled Ca-ATPase, demonstrates that a 2-fold increase in microsecond rotational motion of the Ca-ATPase correlates with the maximal enzymatic activation. Effects of diethyl ether on both the enzymatic activity and molecular dynamics are completely reversible by dilution with buffer. We propose that ether activates by selectively mobilizing lipid chains adjacent to the enzyme, thus facilitating protein motions that are essential for calcium transport.