Spin labeling of calcium-dependent phospholipid-binding proteins

Bovine lung annexins p32 and p34 were spin labeled with an iodoacetamidoproxyl spin label, a reagent that reportedly couples with protein methionine residues. Labeling conditions and stoichiometry were studied with the radiolabeled analogue [1-14C]iodoacetamide. As judged by this method, carboxamidomethylation of both p32 and p34 occurred up to a 0.7 mol ratio after 60 h of reaction at 37 degrees C and at pH 4. The two proteins retained Ca2(+)-dependent phospholipid-binding ability both in radiolabeled and in spin-labeled forms. Electron resonance spectra of spin-labeled p32 and p34 showed the features of a partially immobilized spin probe, with rotational correlation time values of 1.15 and 1.25 ns, respectively, which definitely indicate successful spin labeling. Quantitation of ESR spectra by computer double integration indicated 70% spin labeling of both proteins, as anticipated by radiolabeling. The use of spin-labeled p32 and p34 in the study of Ca2(+)-dependent interaction of annexins with biomembranes is proposed.     

Divalent cation binding to wheat germ calmodulin

The Ca2+ binding to plant (wheat germ) calmodulin was measured in 0.1 M NaCl by a flow-dialysis method. The four macroscopic binding constants best fitted to the data were 0.20, 0.25, 0.025, and 0.0024 microM-1. The cysteine residue of this calmodulin is located at the 27th position from the NH2-terminal (Yazawa, M. et al. (1982) Abstr. 33th Conf. Protein Structure pp. 9-12, Osaka). According to the quantitative analysis of the reaction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) with Cys 27, the calmodulin which binds 3 Ca2+ showed the minimum reactivity with DTNB. This suggests that the site for the third Ca2+ binding is located close to Cys 27. Cys 27 was spin-labeled with N-(2,2,6,6-tetramethyl-4-piperidine-1-oxyl)maleimide, and its ESR spectrum was measured in the presence of Mn2+ and/or Ca2+. The rotational relaxation time of the label (1.2 ns) was increased by about one-tenth with 1 to 2 mol of bound Ca2+, but was unchanged with Mn2+. On the other hand, Mn2+ induced a remarkable quenching of the spectrum. From the decrease in the peak heights of the ESR spectrum, the distance between the label and the first bound Mn2+ was estimated to be 0.8 nm. It is concluded that the first Mn2+ binds to a domain near the NH2-terminal. The difference UV absorption spectrum induced by Mn2+ was similar to that induced by Ca2+. However, the amount of Mn2+ needed to saturate the difference spectrum was 1 mol more than the amount of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

A spin label study of horseradish peroxidase.

The topography of the active sites of native horseradish peroxidase and manganic horseradish peroxidase has been studied with the aid of a spin-labeled analog of benzhydroxamic acid (N-(1-oxyl-2,2,5,5-tetramethylpyrroline-3-carboxy)-p-aminobenzhydroxamic acid). The optical spectra of complexes between the spin-labeled analog of benzhydroxamic acid and Fe3+ or Mn3+ horseradish peroxidase resembled the spectra of the corresponding enzyme complexes with benzhydroxamic acid. Electron spin resonance (ESR) measurement indicated that at pH 7 the nitroxide moiety of the spin-labeled analog of benzhydroxamic acid became strongly immobilized when this label bound to either ferric or manganic horseradish peroxidase. The titration of horseradish peroxidase with the spin-labeled analog of benzhydroxamic acid revealed a single binding site with association constant Ka approximately 4.7 . 10(5) M-1. Since the interaction of ligands (e.g. F-, CN-) and H2O2 with horseradish peroxidase was found to displace the spin label, it was concluded that the spin label did not indeed bind to the active site of horseradish peroxidase. At alkaline pH values, the high spin iron of native horseradish peroxidase is converted to the low spin form and the binding of the spin-labeled analog of benzhydroxamic acid to horseradish peroxidase is completely inhibited. From the changes in the concentration of both bound and free spin label with pH, the pK value of the acid-alkali transition of horseradish peroxidase was found to be 10.5. The 2Tm value of the bound spin label varied inversely with temperature, reaching a value of 68.25 G at 0 degree C and 46.5 G at 52 degrees C. The dipolar interaction between the iron atom and the free radical accounted for a 12% decrease in the ESR signal intensity of the spin label bound to horseradish peroxidase. From this finding, the minimum distance between the iron atom and nitroxide group and hence a lower limit to the depth of the heme pocket of horseradish peroxidase was estimated to be 22 A.     

Antibodies against the 53 kDa glycoprotein inhibit the rotational dynamics of both the 53 kDa glycoprotein and the Ca(2+)-ATPase in the sarcoplasmic reticulum membrane.

The purpose of this study is to better define the relationship of the 53 kDa glycoprotein (GP-53) of the sarcoplasmic reticulum (SR) to other SR proteins. Towards that end the effects of antibodies against GP-53 on the rotational dynamics of maleimide spin-labeled proteins of SR of rabbit skeletal muscle were investigated. The labeling protocol used in this study provided 1.6 +/- 0.3 moles spin label incorporated per 10(5) g SR protein. Labeling specificity studies indicated that nearly 70% of the label bound specifically to the Ca(2+)-ATPase, with the remainder bound to GP-53. Using saturation-transfer electron paramagnetic resonance (ST-EPR), it was determined that the rotational mobility (i.e., the rate of rotation) of the spin-labeled SR proteins decreased greater than 5-fold upon preincubation of MSL-SR with an antiserum against the GP-53, while preincubation of MSL-SR with preimmune serum had no effect. Preincubation of MSL-SR with a monoclonal antibody against the GP-53 produced a 4-fold decrease in the rotational mobility of the MSL-SR proteins compared to control measurements. Further, these effects showed a marked calcium dependence: the decrease in the rotational mobility of the MSL-SR proteins preincubated with anti-GP-53 antibodies in 500 microM Ca2+ was 3-6-fold greater than that of MSL-SR preincubated with antibodies in 5 mM EGTA. While MSL was bound to both Ca(2+)-ATPase and GP-53, model calculations indicated that the decreases observed in the rotational mobility of the MSL-SR proteins caused by the anti-GP-53 monoclonal antibodies were too large to be accounted for by effects on GP-53 alone. The calculations suggest that the rotational rate of Ca(2+)-ATPase was also diminished by anti-GP-53 monoclonal antibodies, indicating an interaction between GP-53 and Ca(2+)-ATPase in the SR membrane.     

Evidence that the two free sulfhydryl groups of plasma fibronectin are in different local environments. Saturation-recovery electron spin resonance study.

Human plasma fibronectin is a dimer consisting of two subunits; each contains two cryptic thiol groups that were selectively labeled with an 15N,2H-maleimide spin label. Previous studies using conventional X-band electron spin resonance (ESR) methods showed that the spectrum of the labeled protein displays a single strongly immobilized component with an effective rotational correlation time of approximately 17 ns, suggesting that the physical environments of the two labeled sites per chain are indistinguishable. Here we have used saturation-recovery ESR to measure directly electron spin-lattice relaxation time (T1) of the labeled protein in solution at 27 degrees C. Interestingly, the time evolution of the signal was found to be biphasic, which was deconvoluted into two T1 values of 1.37 and 4.53 microseconds. Thus, the two spin-labeled sulfhydryl sites of plasma fibronectin (Fn), being similar in rates of rotational diffusion, differ by a factor of 3.2 in T1. Parallel experiments using various fibronectin fragments showed that the 1.37-microseconds component is associated with the label attached onto the thiol located in between the DNA-binding and the cell-binding domains, and the 4.53-microseconds component is associated with the label attached onto the thiol located within the carboxyl-terminal fibrin-binding domain. The data suggest that the saturation-recovery ESR is a useful method for differentiating multiple spin-labeled sites on macromolecules in which the labels undergo similar rates of rotational motion.     

Saturation transfer electron spin resonance of Ca2(+)-ATPase covalently spin-labeled with beta-substituted vinyl ketone- and maleimide-nitroxide derivatives. Effects of segmental motion and labeling levels.

The Ca2(+)-ATPase in native sarcoplasmic reticulum membranes was selectively spin-labeled for saturation transfer electron spin resonance (ESR) studies by prelabeling with N-ethylmaleimide and by using low label/protein ratios. Results with the nitroxide derivative of the standard sulphydryl-modifying reagent, maleimide, were compared with a series of six novel nitroxide beta-substituted vinyl aryl ketone derivatives which differed (with two exceptions) in the substituent at the ketone position. The two exceptions had a different electron withdrawing group at the alpha-carbon, to enhance further the electrophilic character of the beta-carbon. Although differing in their reactivity, all the conjugated unsaturated ketone nitroxide derivatives displayed saturation transfer ESR spectra indicative of much slower motion than did the maleimide derivative. The saturation transfer ESR spectra of maleimide-labeled Ca2(+)-ATPase therefore most likely contain substantial contributions from segmental motion of the labeled group. The effects of the level of spin labeling were also investigated. With increasing degree of spin label incorporation, the linewidths of the conventional ESR spectrum progressively increased and the intensity of the saturation transfer spectrum dropped dramatically, as a result of increasing spin-spin interactions. The hyperfine splittings of the conventional spectrum and the outer lineheight ratios of the saturation transfer spectrum remained relatively unchanged. Extrapolation back to zero labeling level yielded comparable values for the effective rotational correlation times deduced from the saturation transfer spectrum intensities and from the lineheight ratios, for the vinyl ketone label. For the maleimide label the extrapolated values from the integral are significantly lower than those from the lineheight ratios, probably because of the segmental motion. Comparison is made of the effective rotational correlation time for the vinyl ketone label with the predictions of hydrodynamic models for the protein diffusion, in a discussion of the aggregation state of the Ca2(+)-ATPase in the native sarcoplasmic reticulum membrane. The implications for the study of protein rotational diffusion and segmental motion, and of the proximity relationships between labeled groups, using saturation transfer ESR spectroscopy are discussed.     

Study of the binding of substrates and paramagnetic Mn2+ ions with phosphoglycerate kinase by saturating ESR spectra of a spin-labelled protein]

A single SH-group of phosphoglycerate kinase from yeast was modified by mercury-containing spin label. The saturation curves of ESR spectra of the spin-labeled enzyme were studied. The paramagnetic ions of Mn2+ bound to the centre of ion nonspecific binding or active centre in the complex with ATP can influence the saturation of the spin-labeled enzyme. The saturation curves of the ESR signal of the spin-labeled enzyme in the presence of paramagnetic complex of CrATP were studied. It has been demonstrated that the second nonspecific centre of ATP binding is located at the active site of the enzyme (3-phosphoglycerate binding centre).     

Effect of Ca2+ binding on troponin C. Changes in spin label mobility, extrinsic fluorescence, and sulfhydryl reactivity.

The Ca2+ binding component (TnC) of troponin has been selectively labeled with either a spin label, N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) iodoacetamide, or with a fluorescent probe, S-mercuric-N-dansyl cysteine, presumably at its single cysteine residue (Cys-98) in order to probe the interactions of TnC with divalent metals and with other subunits of troponin. The modified protein has the same Ca2+ binding properties as native TnC (Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4628), viz. two Ca2+ binding sites at which Mg2+ appears to compete (Ca2+-Mg2+ sites, KCa = 2 X 10(7) M-1) and two sites at which Mg2+ does not compete (Ca2+-specific sites, KCa = 2 X 10(5) M-1). Either Ca2+ or Mg2+ alters the ESR spectrum of spin-labeled TnC in a manner that indicates a decrease in the mobility of the label, Ca2+ having a slightly greater effect. In systems containing both Ca2+ and Mg2+ the mobility of the spin label is identical with that in systems containing Ca2+ alone. The binding constants for Ca2+ and Mg2+ deduced from ESR spectral changes are 10(7) and 10(3) M-1, respectively, and the apparent affinity for Ca2+ decreases by about an order of magnitude on adding 2 mM Mg2+. Thus, the ESR spectral change is associated with binding of Ca2+ to one or both of the Ca2+-Mg2+ sites. Addition of Ca2+ to the binary complexes of spin-labeled TnC with either troponin T (TnT) or troponin I (TnI) produces greater reduction in the mobility of the spin label than in the case of spin-labeled TnC alone, and in the case of the complex with TnI the affinity for Ca2+ is increased by an order of magnitude. The fluorescence of dansyl (5-dimethylaminonaphthalene-1-sulfonyl)-labeled TnC is enhanced by Ca2+ binding to both high and low affinity sites with apparent binding constants of 2.6 X 10(7) M-1 and 2.9 X 10(5) M-1, respectively, calculated from the transition midpoints. The presence of 2 mM Mg2+, which produces no effect on dansyl fluorescence itself, in contrast to its effect on the spin label, shifts the high affinity constant to 2 X 10(6) M-1. Spectral changes produced by Ca2+ binding to the TnC-TnI complex furnish evidence that the affinity of TnC for Ca2+ is increased in the complex. The reactivity of Cys-98 to the labels and to 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) is decreased by Ca2+ or Mg2+ both with native TnC and in 6 M urea. The reaction rate between Cys-98 and Nbs2 decreases to one-half the maximal value at a Ca2+ concentration that suggests binding to the Ca2+-Mg2+ sites. Formation of a binary complex between TnI and TnC reduces the rate of reaction, and there is a further reduction by Ca2+. The effect of Ca2+ takes place at concentrations that are 1 order of magnitude lower than in the case of TnC alone. These results suggest that the Ca2+ binding site adjacent to Cys-98 is one of the Ca2+-Mg2+ binding sites.     

Electron spin resonance spin label studies of plasma fibronectin: effect of temperature

Plasma fibronectin was chemically modified by 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl (maleimide spin label). Only the free sulfhydryl groups of plasma fibronectin were modified by the label under the experimental conditions. The ESR spectrum of spin-labeled fibronectin showed that the sites of labeling were highly immobilized, suggesting that the sulfhydryl groups of the protein are in small, confined environments. The conversion of the strongly immobilized ESR spectrum into a weakly immobilized one was observed when the spin-labeled protein was heated from 30 to 60 degrees C, indicating the thermal unfolding of the protein molecules. The midpoint temperature for the thermal unfolding of plasma fibronectin is about 50 degrees C. The results suggest that plasma fibronectin is stable to about 40 degrees C and starts unfolding above this temperature. The rotational correlation time estimated from the ESR spectrum of spin-labeled fibronectin at 21 degrees C was about 2.0 X 10(-8) s. The rotational correlation time calculated from the Stokes-Einstein equation, assuming a rigid globular configuration for fibronectin with a Stokes radius of 10 nm, was about 7.8 X 10(-7) s. The differences in rotational correlation time by a factor of 39 between experimental and calculated values do not support a globular configuration for plasma fibronectin.     

Determining the motility of glycoprotein oligosaccharides from lymphocyte membranes using the spin label method

Splenocyte glycoproteins solubilized by papain were purified on lectyl-lectin Sepharose 4B. Glycoproteins eluted from lectin were spin-labeled at carbohydrates. Quantitative evaluation of ESR spectra of spin-labeled glycoproteins pointed to strong restriction of reorientation of the spin label bound to oligosaccharides. Calculated correlation time of relaxing volume tagged with the spin label was equal to the molecular weight about 6000-7000 dalton. This value suggests the existence of flexibility of the glycoproteins studied.     

Heterogeneity in the fluidity of intact erythrocyte membrane and its homogenization upon hemolysis.

Intact erythrocytes were spin-labeled with various classes of phospholipid label. The ESR spectrum for phosphatidylcholine spin label was distinctly different from those for phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidic acid spin labels. The overall splitting for the former (52.5 G) was markedly larger than those for the others (approx. 47 G), suggesting a more rigid phosphatidylcholine bilayer phase and more fluid phosphatidylethanolamine and phosphatidylserine phases in the erythrocyte membrane. Evidence for asymmetric distribution of phospholipids in the membrane was obtained. Spin-labeled phosphatidylcholine incorporated into erythrocytes was reduced immediately by cystein and Fe3+, while the reduction of spin-labeled phosphatidylserine was very slow. The present results therefore suggest asymmetric fluidity in erythrocyte membrane; a more rigid outer layer and a more fluid inner layer. The heterogeneity in the lipid structure was also manifested in the temperature dependence of the fluidity. The overall splitting for phosphatidylcholine spin label showed two inflection points at 18 and 33 degrees C, while that for phosphatidylserine spin label had only one transition at 30 degrees C. When the spin-labeled erythrocytes were hemolyzed, the marked difference in the ESR spectra disappeared, indicating homogenization of the heterogenous fluidity. Mg2+ or Mg2+ + ATP prevented the hemolysis-induced spectral changed. Ca2+ did not prevent the homogenization and acted antagonistically to Mg2+. The heterogeneity preservation by Mg2+ was nullified by trypsin, pronase or N-ethylmaleimide added inside the cell. Some inner proteins may therefore be involved in maintaining the heterogeneous structure. The protecting action of Mg2+ was dependent on hemolysis temperature, starting to decrease at 18 degrees C and vanishing at 40 degrees C. The present study suggests that the heterogeneity in the fluidity of intact erythrocyte membranes arises from interactions between lipids and proteins in the membrane and also from interactions between the membrane constituents and the inner proteins. Concentration of cholesterol in the outer layer may also partly contribute to the heterogeneity.     

Interaction of spin-labeled tryptophan with sickle hemoglobin: probing the microenvironment of the contact sites by spin-probe--spin-label techniques

The spin-labeled tryptophan was used as a structural probe of hemoglobin contact sites. The ESR spectral data indicated that the probe exhibits weak binding to hemoglobin with a dissociation constant of 3.2.10(-5) and 4.0 mol bound per hemoglobin tetramer. The spectrum suggested that the bound tryptophan was 'partially immobilized' with a correlation time reflecting the environment of the tryptophan binding site of 8.2 ns. The topology of the contact sites was investigated by using dual spin-label methodology in which spin-labeled tryptophan and (2H,15N) substituted and deuterated maleimide spin label [2H-15N]MSL covalently-bound to Cys-beta 93 residue were used. The ESR spectral data suggested that the tryptophan binding sites were located within 8-10 A of the nitroxide free radical of spin-labeled hemoglobin. The environment of the contact sites is discussed.     

Ca2+-induced conformational changes of spin-labeled g2 chain bound to myosin and the effect of phosphorylation.

One of the low molecular weight components of myosin, g2, was isolated by alkali treatment of myosin and was chemically modified with a spin label reagent, 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl. The label on g2 showed a rather weakly immobilized ESR spectrum and it was clearly affected by Ca2+; the half-maximal change was at around pCa 4. The spin-labeled g2 was incorporated into myosin by exchange with the intrinsic g2 of myosin in 0.6 M KSCN or 4 M LiC1. The label on g2 became strongly immobilized on association with myosin. Under the conditions used, ESR spectral change due to Ca2+ occurred at two different concentration ranges, which were as low as pCa 8 and at around pCa 4. Phosphorylated g2 was isolated from myosin after the protein kinase [EC 2.1.1.37]-catalyzed phosphorylation of myosin and it was also modified with the maleimide label. Dephosphorylation of the phosphorylated g2 was performed using E. coli alkaline phosphatase [EC 3.1.3.1]. The effects of Ca2+ on the ESR spectra of phosphorylated and dephosphorylated g2 were investigated on the state associated with myosin. A change in the ESR spectrum from strongly immobilized to weakly immobilized states was observed with both g2 chains on the addition of Ca2+. However, the effective concentration ranges of Ca2+ were quite different; around pCa 4 for the phosphorylated g2 and around pCa 8 for the dephosphorylated g2. The results indicate that g2 undergoes a conformational change at physiological levels of Ca2+ sufficient to saturate troponin, but it does not do so after phosphorylation.     

Spin-labeling of adenosine triphosphatase in sarcoplasmic reticulum membrane and change in the state of the spin labels induced by deoxycholate.

Fragmented sarcoplasmic reticulum (SR) was reacted with a thiol-directed spin label, N-(1-oxyl-2,2,6,6,-tetramethyl-4-piperidinyl)maleimide, under various conditions. It was found that ATP inhibited the binding of the label to SR protein in the initial phase of the reaction, but as the incubation time was extended up to 18 h, the amount of label bound to SR protein in the control and ATP-containing samples became almost identical. The Ca2+-dependent ATPase control and ATP-containing samples became almost identical The Ca2+-dependent ATPase (ATP phosphohydrolase [EC 3.6.1.3]) of SR was protected by the presence of ATP during incubation with relatively low concentrations of spin label, irrespective of the total amount of label bound, although with increasing concentration of bound label the ATPase activity decreased. Deoxycholate slightly reduced the rotational freedom of the label bound to SR protein and decreased the initial rate of quenching of protein-bound nitroxide by ascorbate. From an analysis of these results, it was concluded that the binding of deoxycholate to protein decreases the accessibility of ascorbate to the protein-bound label.     

Conformation of spin-labeled tropomyosin in reconstituted muscle thin filaments in response to calcium ion and heavy meromyosin

Tropomyosin (TM) exists in thermal equilibrium between a highly structured N state, a partially unfolded X state, and a completely unfolded D state, i.e., N in equilibrium X in equilibrium D. The strongly immobilized electron spin resonance (ESR) spectral component of spin-labeled TM corresponds to TM in the N state and the weakly immobilized component to TM in the X state below the main unfolding transition and to TM in the D state above this transition [Graceffa, P., & Lehrer, S. S. (1984) Biochemistry 23, 2606-2612]. The addition of actin, troponin (TN), and heavy meromyosin (HMM) to spin-labeled TM reduces the ratio of weakly to strongly immobilized labels, indicating a shift in the N in equilibrium X in equilibrium D equilibrium toward the N state. At 37 degrees C, for spin-labeled TM alone K (=X/N) greater than 1.0 with some TM in the D state, K = 0.8 for spin-labeled TM bound to actin, and K less than 0.05 for spin-labeled TM bound to actin + TN +/- Ca2+, actin + HMM + TN +/- Ca2+, and actin + HMM. Thus, actin + TN dramatically shifts the TM structure to the N conformation with little further effect upon addition of Ca2+ or HMM. The temperature at which spin-labeled TM begins to dissociate from a protein complex was determined from the temperature dependence of the ESR spectra.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rotational dynamics of spin-labeled F-actin during activation of myosin S1 ATPase using caged ATP.

The most probable source of force generation in muscle fibers in the rotation of the myosin head when bound to actin. This laboratory has demonstrated that ATP induces microsecond rotational motions of spin-labeled myosin heads bound to actin (Berger, C. L. E. C. Svensson, and D. D. Thomas. 1989. Proc. Natl. Acad. Sci. USA. 86:8753-8757). Our goal is to determine whether the observed ATP-induced rotational motions of actin-bound heads are accompanied by changes in actin rotational motions. We have used saturation transfer electron paramagnetic resonance (ST-EPR) and laser-induced photolysis of caged ATP to monitor changes in the microsecond rotational dynamics of spin-labeled F-actin in the presence of myosin subfragment-1 (S1). A maleimide spin label was attached selectively to cys-374 on actin. In the absence of ATP (with or without caged ATP), the ST-EPR spectrum (corresponding to an effective rotational time of approximately 150 microseconds) was essentially the same as observed for the same spin label bound to cys-707 (SH1) on S1, indicating that S1 is rigidly bound to actin in rigor.(ABSTRACT TRUNCATED AT 250 WORDS)     

ESR studies of the erythrocyte membrane skeletal protein network: influence of the state of aggregation of spectrin on the physical state of membrane proteins, bilayer lipids, and cell surface carbohydrates

The stability of the human erythrocyte membrane skeletal network is reported to be dependent on the state of aggregation of spectrin and decreased or increased by polyphosphate anions or the polyamine, spermine, respectively. We have employed polyacrylamide gel electrophoresis and electron spin resonance (ESR) utilizing spin labels specific for membrane proteins, bilayer lipids, or cell-surface sialic acid in order to gain insight into these observations and into the reliability of the ESR spectra of the protein-specific spin label used to correctly report the interactions of the skeletal protein network. The major findings are: (1) We confirm previous reports that the preferred state of spectrin aggregation in the skeletal network is tetrameric and that spectrin can be reversibly transformed to dimeric spectrin and back to tetrameric spectrin on the membrane. (2) The ESR spectra of the protein specific maleimide spin label employed accurately reflect the state of aggregation of spectrin. (3) As dimeric spectrin is increased on the membrane or when 2,3-bisphosphoglycerate was added to spin-labeled membranes, increased segmental motion of protein spin label binding sites reflecting decreased protein-protein interactions in the skeletal network is observed (P less than 0.002 and P less than 0.005, respectively). (4) Conversely, as protein-protein interactions between skeletal proteins or between skeletal proteins and the bilayer are increased by spermine (reflected in the total inability to extract spectrin from the membrane in contrast to control membranes), highly decreased segmental motion of the protein specific spin label binding site is observed (P less than 0.005). (5) The dimeric-tetrameric state of spectrin aggregation on the membrane does not have influence on the order or motion of bilayer lipids nor on the rotational rate of spin-labeled, cell-surface sialic acid, a result also observed when protein-protein interactions were decreased by 2,3-bisphosphoglycerate. In contrast, increased protein-protein interactions by addition of spermine produced a small, but significant, increase in order and decrease in motion of bilayer lipids near the membrane surface as well as a nearly 40% decrease in the apparent rotational correlation time of spin labeled, cell surface sialic acid (P less than 0.002). These latter observations are discussed with reference to possible associations of phospholipids and the major, transmembrane sialoglycoprotein with the skeletal protein network.     

Low microwave-amplitude ESR spectroscopy: measuring spin-relaxation interactions of moderately immobilized spin labels in proteins

Electron spin resonance (ESR) spectroscopy in combination with site-directed spin labeling (SDSL) is a powerful tool for determining protein structure, dynamics and interactions. We report here a method for determining interactions between spin labels and paramagnetic relaxation agents, which is performed under subsaturating conditions. The low microwave-field amplitude employed (h(1)<0.36 G) only requires standard, commercially available ESR equipment. The effect of relaxation enhancement on the spin-spin-relaxation time, T(2e), is measured by this method, and compared to classical progressive power saturation performed on a free spin label, (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl)methanethiosulfonate (MTSL), and a spin-labeled protein (Thermomyces lanuginosa lipase, TLL-I252C), employing the water-soluble relaxation agent chromium(III) oxalate (Crox) in concentrations between 0-10 mM. The low-amplitude theory showed excellent agreement with that of classical power saturation in quantifying Crox-induced relaxation enhancement. Low-amplitude measurements were then performed using a standard resonator, with Crox, on 11 spin-labeled TLL mutants displaying rotational correlation times in the motional narrowing regime. All spin-labeled proteins exhibited significant changes in T(2e). We postulate that this novel method is especially suitable for studying moderately immobilized spin labels, such as those positioned at exposed sites in a protein. This method should prove useful for research groups with access to any ESR instrumentation.     

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.     

Interaction of spin-labeled tryptophan with sickle hemoglobin: probing the microenvironment of the contact sites by spin-probe-spin-label techniques

The spin-labeled tryptophan was used as a structural probe of hemoglobin contact sites. The ESR spectral data indicated that the probe exhibits weak binding to hemoglobin with a dissociation constant of 3.2 · 10⁻⁵ and 4.0 mol bound per hemoglobin tetramer. The spectrum suggested that the bound tryptophan was ‘partially immobilized’ with a correlation time reflecting the environment of the tryptophan binding site of 8.2 ns. The topology of the contact sites was investigated by using dual spin-label methodology in which spin-labeled tryptophan and (²H,¹⁵N) substituted and deuterated maleimide spin label [²H-¹⁵N]MSL covalently-bound to Cys-ß93 residue were used. The ESR spectral data suggested that the tryptophan binding sites were located within 8–10Åof the nitroxide free radical of spin-labeled hemoglobin. The environment of the contact sites is discussed.