The binding isotherms for the interaction of 5-doxyl stearic acid with bovine and human albumin.

Binding isotherms for the interaction of 5-doxyl stearic acid with bovine and human albumin are reported. The critical micelle concentration (CMC) and the limiting solubility of 5-doxyl stearic acid were determined using the electron spin resonance (ESR)-spin label method. The CMC and the limiting solubility of this spin-label stearic acid in saline-phosphate buffer are 3.5 x 10(-5) M and 2 x 10(-4) M, respectively. We found no ESR line width evidence for pre-association of the spin-label stearate below the CMC. Maximum binding of the spin-label stearate to both bovine and human albumin occurs before micelle formation. The binding isotherm for spin-label stearic acid interaction with bovine albumin is in agreement with data obtained by others using [1-(14)C]stearic acid. For human albumin, comparison is difficult since previous data obtained with [1-(14)C]stearic acid vary widely. Comparison of the ESR 2T(||) values (the splitting between low and high field extremes, a measure of the degree of immobilization of protein-bound spin-label stearate) for bovine and human albumin indicates a greater immobilization of the spin-label molecules bound to human albumin. The binding data indicate that complexes are formed with bound spin-label stearate/albumin ratios of at least 18. The computed equilibrium constants for both bovine and human albumin indicate that the first seven spin-label molecules are tightly bound, log K > 5.0. The species predicted to form in solution by these equilibrium constants are reported.     

Specific surface association of avidin with N-biotinylphosphatidylethanolamine membrane assemblies: effect on lipid phase behavior and acyl-chain dynamics.

The interaction of avidin with aqueous dispersions of N-biotinylphosphatidylethanolamines, of acyl chain lengths C(14:0), C(16:0), and C(18:0), was studied by using spin-label electron spin resonance (ESR) spectroscopy, (31)P nuclear magnetic resonance ((31)P NMR) spectroscopy, differential scanning calorimetry, and chemical binding assays. In neutral buffer containing 1 M NaCl, binding of avidin is due to specific interaction with the biotinyl lipid headgroup because avidin presaturated with biotin does not bind. Saturation binding of the protein corresponds to a ratio of 50 lipid molecules per tetrameric avidin. Phospholipid probes spin-labeled at various positions between C-4 and C-14 in the sn-2 chain were used to characterize the effects of avidin binding on the lipid chain dynamics. In the fluid phase, protein binding results in a decrease of chain mobility at all positions of labeling while the flexibility gradient characteristic of a liquid-crystalline lipid phase is maintained. There is no evidence from the spin-label ESR spectra for penetration of the protein into the hydrophobic interior of the membrane. At temperatures corresponding to the gel phase, the lipid chain mobility increases on binding protein. The near constancy in mobility found with chain position, however, suggests that in the gel phase the lipid chains remain interdigitated upon binding avidin. Binding of increasing amounts of avidin results in a gradual decrease of the lipid chain-melting transition enthalpy with only small change in the transition temperature. At saturation binding, the calorimetric enthalpy is reduced to zero. (31)P NMR spectroscopy indicates that protein binding increases the surface curvature of dispersions of all three biotin lipids. The C(14:0) biotin lipid yields isotropic (31)P NMR spectra in the presence of avidin at all temperatures between 10 and 70 degrees C, in contrast to dispersions of the lipid alone, which give lamellar spectra at low temperature that become isotropic at the chain-melting temperature. In the presence of avidin, the C(16:0) and C(18:0) biotin lipids yield primarily lamellar (31)P NMR spectra at low temperature with a small isotropic component; the intensity of the isotropic component increases with temperature, and the spectra narrow and become totally isotropic at high temperature, in contrast to dispersions of the lipids alone, which give lamellar spectra in the fluid phase. The binding of avidin therefore reduces the cooperativity of the biotin lipid packing, regulates the mobility of the lipid chains, and enhances the surface curvature of the lipid aggregates. These effects may be important for both lateral and transbilayer communication in the membrane.     

Novel spin-labels for the study of lipid-protein interactions. Application to (Na+, K+)-ATPase membranes

The interactions of a series of spin-labeled fatty acids, in which the nitroxide ring is incorporated in different ways as an integral part of the hydrocarbon chain, with the (Na+,K+)-ATPase in membranes from Squalus acanthias, have been studied by electron spin resonance spectroscopy. The fatty acids are 2,4-, 2,5-, and 3,2-substituents of 2,2,5,5-tetramethylpyrrolidine-N-oxyl and belong to the class of minimal perturbation nitroxide probes. For all five fatty acid labels, a motionally restricted lipid component was observed in the ESR spectra of (Na+,K+)-ATPase membranes, in addition to the fluid component, which was found in the spectra of the extracted membrane lipids. The pH dependence of the motionally restricted spin-label population indicated a sensitivity in the selectivity of the lipid-protein interaction to the protonation state of the fatty acid. These results agree with those found previously for the conventional oxazolidine (doxyl) fatty acid and phospholipid spin-label derivatives [Esmann, M., Watts, A., & Marsh, D. (1985) Biochemistry 24, 1386-1393] and indicate that the motion of the lipid chains is significantly hindered by interaction with the protein, irrespective of the nature of the spin-label group.     

Time-resolved electron spin resonance studies of spin-labelled lipids in membranes

Recently, developments in time-resolved spin-label electron spin resonance (ESR) spectroscopy have contributed considerably to the study of biomembranes. Two different applications of electron spin echo spectroscopy of spin-labelled phospholipids are reviewed here: (1) the use of partially relaxed echo-detected ESR spectra to study the librational lipid-chain motions in the low-temperature phases of phospholipid bilayers; (2) the use of electron spin echo envelope modulation spectroscopy to determine the penetration of water into phospholipid membranes. Results are described for phosphatidylcholine bilayer membranes, with and without equimolar cholesterol, that are obtained with phosphatidylcholine spin probes site-specifically labelled throughout the sn-2 chain.     

Rotational mobility and orientational stability of a transport protein in lipid membranes

A single-cysteine mutant of the lactose transport protein LacS(C320A/W399C) from Streptococcus thermophilus was selectively labeled with a nitroxide spin label, and its mobility in lipid membranes was studied as a function of its concentration in the membrane by saturation-transfer electron spin resonance. Bovine rhodopsin was also selectively spin-labeled and studied to aid the interpretation of the measurements. Observations of spin-labeled proteins in macroscopically aligned bilayers indicated that the spin label tends to orient so as to reflect the transmembrane orientation of the protein. Rotational correlation times of 1-2 micros for purified spin-labeled bovine rhodopsin in lipid membranes led to viscosities of 2.2 poise for bilayers of dimyristoylphosphatidylcholine (28 degrees C) and 3.0 poise for the specific mixture of lipids used to reconstitute LacS (30 degrees C). The rotational correlation time for LacS did not vary significantly over the range of low concentrations in lipid bilayers, where optimal activity was seen to decrease sharply and was determined to be 9 +/- 1 micros (mean +/- SD) for these samples. This mobility was interpreted as being too low for a monomer but could correspond to a dimer if the protein self-associates into an elongated configuration within the membrane. Rather than changing its oligomeric state, LacS appeared to become less ordered at the concentrations in aligned membranes exceeding 1:100 (w/w) with respect to the lipid.     

Weak substrate binding to transport proteins studied by NMR.

The weak binding of sugar substrates fails to induce any quantifiable physical changes in the L-fucose-H+ symport protein, FucP, from Escherichia coli, and this protein lacks any strongly binding ligands for competitive binding assays. Access to substrate binding behavior is however possible using NMR methods which rely on substrate immobiliza-tion for detection. Cross-polarization from proton to carbon spins could detect the portion of 13C-labeled substrate associated with 0.2 micromol of the functional transport system overexpressed in the native membranes. The detected substrate was shown to be in the FucP binding site because its signal was diminished by the unlabeled substrates L-fucose and L-galactose but was unaffected by a three- to fivefold molar excess of the non-transportable stereoisomer D-fucose. FucP appeared to bind both anomers of its substrates equally well. An NMR method, designed to measure the rate of substrate exchange, could show that substrate exchanged slowly with the carrier center (>10(-1) s), although its dynamics are not necessarily coupled strongly to this site within the protein. Relaxation measurements support this view that fluctuations in the interaction with substrate would be confined to the binding site in this transport system.     

Spin-label studies on the origin of the specificity of lipid-protein interactions in Na+,K+-ATPase membranes from Squalus acanthias

The pH dependence and salt dependence of the lipid-protein interactions of phosphatidic acid, phosphatidylserine, and stearic acid with Na+,K+-ATPase membranes from Squalus acanthias have been studied with spin-label electron spin resonance spectroscopy, using lipids with nitroxide labels on the 14-position C atom of the sn-2 chain. For phosphatidic acid and stearic acid, the fraction of motionally restricted spin-label increases with increasing pH, with pKa's of 6.6 and 8.0, respectively. In contrast, the pKa of stearic acid in the bulk lipid environment of the membrane is estimated from spin-label spectroscopy to be approximately equal to 6.6. The fraction of motionally restricted phosphatidylserine spin-label remains constant over the pH range 4.7-9.2. In the fully dissociated state the fractions of motionally restricted spin-labeled phosphatidic and stearic acids decrease with increasing salt concentration, reaching an approximately constant value at [NaCl] = 0.5-1.0 M. For stearic acid the net decrease is comparable to that obtained on protonation, but for phosphatidic acid the decrease is considerably smaller (by approximately 55%) than that obtained on protonating the lipid. The fraction of motionally restricted phosphatidylserine spin-label varies relatively little with salt concentration up to 1 M NaCl. Direct electrostatic effects alone cannot account for the whole of the observed specificity of interaction of the two phospholipids with Na+,K+-ATPase membranes.     

Involvement of the 3' side of the anticodon loop of yeast tRNATyr in messenger-free binding to ribosomes. An electron-spin resonance study

Electron-spin resonance (ESR) spectra of a nitroxide spin-label attached to residue i6A-37 of yeast tRNATyr were measured in complexes of deacylated tRNATyr with Escherichia coli ribosomes. A Scatchard plot, obtained in the absence of mRNA, indicated strong binding with an association constant of 1 X 10(7) l X mol-1, suggesting the P-site binding. The ESR spectrum of free tRNATyr, characteristic for a rapidly tumbling nitroxide, changes to a spectrum with extensively broadened lines in the ribosome-tRNA complex. The original spectrum can be restored upon long incubations of the complex with an excess of extraneous tRNA. ESR spectra suggest that the spin-label motion is drastically perturbed though not completely blocked in the ribosome-tRNATyr complex. Since ESR spectra of a spin-label attached to the opposite, i.e. 5', side of the anticodon loop are only slightly perturbed by the messenger-free binding to ribosomes [Rodriguez et al. (1980) J. Biol. Chem. 255, 8116-8120], it is concluded that the two sides of the anticodon loop face entirely different environments when bound to the P site, the 3' side being oriented towards the surface of the ribosome, and the other side towards its environment or a large cavity.     

Methionine-90-spin-labeled bovine alpha-lactalbumin: electron spin resonance and NMR distance measurements

The unique methionine residue of bovine alpha-lactalbumin was modified by irreversible alkylation with the bromoacetamido nitroxide spin-label 4-(2-bromoacetamido)-2,2,6,6-tetramethylpiperidine-N-oxyl. The line shape of the electron spin resonance (ESR) spectrum was indicative of a fairly mobile spin-label and was sensitive to the calcium-induced conformational change. Paramagnetic broadening of the spin-label ESR lines by a Gd(III) ion substituted at the high-affinity calcium site of the protein yielded a distance between the spin-label and the metal-binding site of 8.0 +/- 1.0 A. The extent of the paramagnetic line broadening by the covalently attached nitroxide spin-label on the proton resonances of several amino acid residues of the protein at 500 MHz allowed estimation of intramolecular distances between the methionine-90 residue and several resolvable protons.     

Probing the topography of lectins with site-specific spin-labeled glycosides

Three new spin-labeled glycosides, spin-label I [1-[4-(beta-D-galactopyranosyloxy)phenyl]-3-(2,2,6,6-tetramethyl-1 -oxypiperidin-4-yl)-2-thiourea], spin-label II (2,2,6,6-tetramethyl-1-oxypiperidin-4-yl alpha-D-galactopyranoside), and spin-label III [1-(methyl 2-deoxy-alpha-D-galactopyranosid-2-yl)-3-(2,2,6,6- tetramethyl-1-oxypiperidin-4-yl)-2-thiourea], were investigated as structural probes of Griffonia simplicifolia I isolectins (GS I) A4 and B4, respectively, by electron spin resonance (ESR) and inhibition of guaran isolectin precipitation. The p-aminophenyl beta-galactoside spin-label I was strongly immobilized by the B4 isolectin (Kd = 0.42 mM; 2T parallel = 54.0 +/- 0.3 G), while binding to the A4 isolectin was so weak (KI congruent to 2 mM) that binding was undetectable by ESR. The preference for the B4 isolectin was indicative of a more extended hydrophobic binding locus adjacent to the carbohydrate-specific binding site. The alpha-galactosyl spin-label II bound slightly more strongly to the A4 than to the B4 isolectin, as evidenced in both Kd values and particularly by differences in the degree of immobilization (2T parallel = 53.5 vs. 51.5 G, respectively). The 2-N-substituted methyl galactoside spin-label III was so poor an inhibitor of both isolectins (KI congruent to 1-2 mM) that ESR detection of the bound complex was not feasible. In all cases above, the spin-labels were displaced by specific monosaccharide haptens.     

Binding of the three-repeat domain of tau to phospholipid membranes induces an aggregated-like state of the protein

In patients with Alzheimer's disease, the microtubule-associated protein tau is found aggregated into paired helical filaments (PHFs) in neurofibrillary deposits. In solution, tau is intrinsically unstructured. However, the tubulin binding domain consisting of three or four 31-32 amino acid repeat regions exhibits both helical and β-structure propensity and makes up the proteolysis resistant core of PHFs. Here, we studied the structure and dynamics of the three-repeat domain of tau (i.e. K19) when bound to membranes consisting of a phosphatidylcholine and phosphatidylserine mixture or phosphatidylserine alone. Tau K19 binds to phospholipid vesicles with submicromolar affinity as measured by fluorescence spectroscopy. The interaction is driven by electrostatic forces between the positively charged protein and the phospholipid head groups. The structure of the membrane-bound state of K19 was studied using CD spectroscopy and solid-state magic-angle spinning NMR spectroscopy. To this end, the protein was selectively (13)C-labeled at all valine and leucine residues. Isotropic chemical shift values of tau K19 were consistent with a β-structure. In addition, motionally averaged (1)H-(13)C dipolar couplings indicated a high rigidity of the protein backbone. The structure formation of K19 was also shown to depend on the charge density of the membrane. Phosphatidylserine membranes induced a gain in the α-helix structure along with an immersion of K19 into the phospholipid bilayer as indicated by a reduction of the lipid chain (2)H NMR order parameter. Our results provide structural insights into the membrane-bound state of tau K19 and support a potential role of phospholipid membranes in mediating the physiological and pathological functions of tau.     

Lipid mobility and order in bovine rod outer segment disk membranes. A spin-label study of lipid-protein interactions

Lipid-protein interactions in bovine rod outer segment disk membranes have been studied by using a series of eight stearic acid spin-label probes which were labeled at different carbon atom positions in the chain. In randomly oriented membrane dispersions, the electron spin resonance (ESR) spectra of the C-8, C-9, C-10, C-11, C-12, C-13, and C-14 atom positional isomers all apparently consist of two components. One of the components corresponds closely to the spectra obtained from dispersions of the extracted membrane lipids, and the other, which is characterized by a considerably greater degree of motional restriction of the lipid chains, is induced by the presence of the protein. Digital subtraction has been used to separate the two components. The proportion of the motionally restricted lipid component is approximately constant, independent of the position of the spin-label group, and corresponds to 30-40% of the total spin-label spectral intensity. The hyperfine splitting of the outer maxima in the difference spectra of the motionally restricted component decreases, and concomitantly, the line widths increase with increasing temperature but change relatively little with increasing distance of the spin-label group from the polar head-group region. This indicates that the corresponding chain motions of the protein-interacting lipids lie in the slow-motion regime of spin-label ESR spectroscopy (tau R approximately 10(-8) S) and that the mobility of these lipids increases with increasing temperature but does not vary greatly along the length of the chain. The data from the hyperfine splittings also suggest the existence of a polarity gradient immediately adjacent to the protein surface, as observed in the fluid lipid regions of the membrane. The more fluid lipid component is only slightly perturbed relative to the lipids alone (for label positions 5-14, inclusive), indicating the presence of chain motions on the nanosecond time scale, and the spectra also reveal a similar polarity profile in both lipid and membrane environments. ESR spectra have also been obtained as a function of magnetic field orientation with oriented membrane samples. For the C-14 atom positional isomer, the motionally restricted component is observed to have a large hyperfine splitting, with the magnetic field oriented both parallel and perpendicular to the membrane normal. This indicates that the motionally restricted lipid chains have a broad distribution of orientations at this label position.(ABSTRACT TRUNCATED AT 400 WORDS)     

The key residue for substrate transport (Glu14) in the EmrE dimer is asymmetric

Transport proteins exhibiting broad substrate specificities are major determinants for the phenomenon of multidrug resistance. The Escherichia coli multidrug transporter EmrE, a 4-transmembrane, helical 12-kDa membrane protein, forms a functional dimer to transport a diverse array of aromatic, positively charged substrates in a proton/drug antiport fashion. Here, we report (13)C chemical shifts of the essential residue Glu(14) within the binding pocket. To ensure a native environment, EmrE was reconstituted into E. coli lipids. Experiments were carried out using one- and two-dimensional double quantum filtered (13)C solid state NMR. For an unambiguous assignment of Glu(14), an E25A mutation was introduced to create a single glutamate mutant. Glu(14) was (13)C-labeled using cell-free expression. Purity, labeling, homogeneity, and functionality were probed by mass spectrometry, NMR spectroscopy, freeze fracture electron microscopy, and transport assays. For Glu(14), two distinct sets of chemical shifts were observed that indicates structural asymmetry in the binding pocket of homodimeric EmrE. Upon addition of ethidium bromide, chemical shift changes and altered line shapes were observed, demonstrating substrate coordination by both Glu(14) in the dimer.     

Time-resolved site-directed spin-labeling studies of bacteriorhodopsin: loop-specific conformational changes in M

A spin-label at site 101 in the C-D loop of bacteriorhodopsin was previously found to detect a conformational change during the M --> N transition [Steinhoff, H. -J., Mollaaghababa, R., Altenbach, C., Hideg, K., Krebs, M. P., Khorana, H. G., and Hubbell, W. L. (1994) Science 266, 105-107]. We have extended these time-resolved electron paramagnetic resonance studies in purple membranes by analyzing conformational changes detected by a spin-label at another site in the C-D loop (103), and at sites in the A-B loop (35), the D-E loop (130), and the E-F loop (160). In addition, we have investigated the motion detected by a spin-label at site 101 in a D96A mutant background that has a prolonged M intermediate. We find that among the examined sites, only spin-labels in the C-D loop detect a significant change in the local environment after the rise of M. Although the D96A mutation dramatically prolongs the lifetime of the M intermediate, it does not perturb either the structure of bacteriorhodopsin or the nature of the light-activated conformational change detected by a spin-label at site 101. In this mutant, a conformational change is detected during the lifetime of M, when no change in the 410 nm absorbance is observed. These results provide direct structural evidence for the heterogeneity of the M population in real time, and demonstrate that the motion detected at site 101 occurs in M, prior to Schiff base reprotonation.     

A structure-based simulation approach for electron paramagnetic resonance spectra using molecular and stochastic dynamics simulations

Electron paramagnetic resonance (EPR) spectroscopy using site-directed spin-labeling is an appropriate technique to analyze the structure and dynamics of flexible protein regions as well as protein-protein interactions under native conditions. The analysis of a set of protein mutants with consecutive spin-label positions leads to the identification of secondary and tertiary structure elements. In the first place, continuous-wave EPR spectra reflect the motional freedom of the spin-label specifically linked to a desired site within the protein. EPR spectra calculations based on molecular dynamics (MD) and stochastic dynamics simulations facilitate verification or refinement of predicted computer-aided models of local protein conformations. The presented spectra simulation algorithm implies a specialized in vacuo MD simulation at 600 K with additional restrictions to sample the entire accessible space of the bound spin-label without large temporal effort. It is shown that the distribution of spin-label orientations obtained from such MD simulations at 600 K agrees well with the extrapolated motion behavior during a long timescale MD at 300 K with explicit water. The following potential-dependent stochastic dynamics simulation combines the MD data about the site-specific orientation probabilities of the spin-label with a realistic rotational diffusion coefficient yielding a set of trajectories, each more than 700 ns long, essential to calculate the EPR spectrum. Analyses of a structural model of the loop between helices E and F of bacteriorhodopsin are illustrated to demonstrate the applicability and potentials of the reported simulation approach. Furthermore, effects on the motional freedom of bound spin-labels induced by solubilization of bacteriorhodopsin with Triton X-100 are examined.     

Selective degradation with phospholipases D and C of radioactive isomeric spin-labelled lipids bound to guinea pig liver microsomal membranes.

Membrane-bound lipids of isolated guinea pig liver microsomal membranes were selectively enzymatically labelled with isomeric (5-, 12-, and 16-)doxyl stearic acid. After reisolation, the membranes were degraded with phospholipases D and C under conditions not requiring detergents or organic solvent activators. The degradation of membrane-bound lipids occurred according to the recognized specificity of phospholipases D and C. Temperature-induced changes of degraded membranes containing radioactive spin-labelled isomeric lipids were followed by the electron spin resonance and spectral changes correlated with the lipid composition of membranes. Discontinuities in plots of experimental spectral parameters versus temperature detected in the case of microsomal membranes before and after degradation with phospholipases D and C were attributed to lipid-protein and lipid-lipid interaction(s). On the basis of these and control experiments, discontinuity at around 10-12 degrees C was attributed to the microsomal membrane phosphatidylcholine intrinsic microsomal membrane protein interaction(s), while discontinuities detected at 19-21 degrees C approximately and at 20-30 degrees C approximately were attributed to the phase separation of Ca or Zn salts of membranous phosphatidic acid and to the similar phenomenon involving membrane-bound diglycerides respectively.     

Structure and flexibility of plasma fibronectin in solution: electron spin resonance spin-label, circular dichroism, and sedimentation studies

Human plasma fibronectin has been investigated by electron spin resonance (ESR) spin-label methods in conjunction with circular dichroism (CD) and sedimentation techniques to investigate its structure and flexibility in solution. The buried sulfhydryl groups of fibronectin were modified with a maleimide spin-label [Lai, C.-S., & Tooney, N. M. (1984) Arch. Biochem. Biophys. 228, 465-473]. Both conventional and saturation transfer ESR spectra give a rotational correlation time of about (2-3) X 10(-8) s for plasma fibronectin, a value that is at least 40 times faster than the rotational correlation time calculated from the minimal molecular dimensions. This argues that plasma fibronectin is not a compact, globular protein and suggests that the regions of ordered structural domains have a relatively high degree of independent mobility. ESR, CD, and sedimentation measurements showed that many structural features of plasma fibronectin remain unchanged when the pH is decreased from 7.4 to 3.0. On the other hand, ESR results indicate an unfolding of the protein molecule either at pH 11 or in 4 M urea solution. Similarly, the sedimentation coefficient decreases from about 13 to 8.4 S when the pH is raised to 10.8. At pH values above 11, the CD spectrum resembles a random coil; however, some ordered structure is retained either at pH 11 or in 4 M urea. It is likely that the sulfhydryl-containing regions of the molecule are more sensitive to urea or alkali than are portions of the molecule stabilized by intrachain disulfide bonds.(ABSTRACT TRUNCATED AT 250 WORDS)     

An ESR study of spin-labeled silk fibroin membranes and spin-labeled glucose oxidase immobilized in silk fibroin membranes

This article describes the characteristics of silk fibroin membranes and glucose oxidase, immobilized in membranes as determined by a variety of physical methods, mainly the spin-label electron spin resonance (ESR) method. The properties of membranes insolubilized by different methods, i. e., immersion in 80% methanol aqueous solution, uniaxially drawing by placing on a stretcher, and hydration by placing in a desiccator of 96% relative humidity (RH) for 17 h, are compared. The results are also analyzed in relation to ESR spectra of spin-labeled immobilized glucose oxidase and 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy as a model of the substrate. It is concluded that the heterogeneous structures of the swollen membranes in water differ locally among membranes insolubilized by different methods, but the immobilized state of the enzyme in such membranes is mostly similar. This is correlated to the fact that the thermal or pH stabilities are essentially same among glucose-oxidase-immobilized silk fibroin membranes insolubilized by different methods.     

Preparation and characterization of spin-labeled derivatives of epidermal growth factor (EGF) for investigations of the interactions of EGF with its receptor by electron paramagnetic resonance spectroscopy.

We prepared, purified, and characterized derivatives of epidermal growth factor (EGF) having a nitroxide spin-label attached covalently at the amino terminus. Characterization of these derivatives with regard to the positions of attachment of the spin-label was accomplished by a combination of peptide mapping, protein sequencing, and fast atom bombardment-mass spectrometry. One derivative was chosen for use in initial investigations by electron paramagnetic resonance (EPR) spectroscopy of receptor-bound EGF and its dissociation kinetics. This derivative was found to be equipotent with the native hormone in competitive binding assays, in activating the EGF receptor kinase, and in stimulating the formation of EGF receptor dimers in solubilized cell extracts. Upon binding to solubilized EGF receptor, the spin-labeled EGF derivative became immobilized, giving rise to a visually distinct slow-motion EPR spectrum. The resulting spectrum showed no detectable dipolar interaction between nitroxides, indicating that the nitroxide moieties of spin-labels reacted at the amino termini of receptor-bound spin-labeled EGF molecules are separated by a distance of at least 16 A. An EPR study of the kinetics of dissociation of spin-labeled EGF in the presence of excess unlabeled EGF revealed a rapid component with a k off approximately 2 x 10(-2) s-1 and a less well resolved slow component.     

Conformation of a peptide encompassing the proton translocation channel of vacuolar H(+)-ATPase

The structural properties of a crucial transmembrane helix for proton translocation in vacuolar ATPase are studied using double site-directed spin-labeling combined with electron spin resonance (ESR) (or electron paramagnetic resonance) and circular dichroism spectroscopy in sodium dodecyl sulfate micelles. For this purpose, we use a synthetic peptide derived from transmembrane helix 7 of subunit a from the yeast Saccharomyces cerevisiae vacuolar proton-translocating ATPase that contains two natural cysteine residues suitable for spin-labeling. The interspin distance is calculated using a second-moment analysis of the methanethiosulfonate spin-label ESR spectra at 150 K. Molecular dynamics simulation is used to study the effect of the side-chain dynamics and backbone dynamics on the interspin distance. Based on the combined results from ESR, circular dichroism, and molecular dynamics simulation we conclude that the peptide forms a dynamic alpha-helix. We discuss this finding in the light of current models for proton translocation. A novel role for a buried charged residue (H729) is proposed.