The rotational diffusion of chloroplast phosphate translocator and of lipid molecules in bilayer membranes

The rotational mobility of the phosphate translocator from the chloroplast envelope and of lipid molecules in the membrane of unilamellar azolectin liposomes has been investigated. The rotational dynamics of the liposome membrane were investigated by measuring the rotational diffusion of eosin-5-isothiocyanate(EITC)-labeled L-alpha-dipalmitoylglycerophosphoethanolamine (Pam2 GroPEtn) in the lipid phase of the vesicles, either in the presence or absence of the reconstituted phosphate translocator. The temperature dependence of the anisotropy decay showed that above 25 degrees C the main contribution to the anisotropy decay was caused by uniaxial anisotropic rotation of the labelled lipid molecules around the axis normal to the membrane plane. The rate of rotation of the labelled lipid molecules was strongly dependent on the viscosity of the medium (eta 1). Extrapolation to eta 1 = 0 Pa.s yielded a correlation time of phi = 20 +/- 5 ns, t = 30 degrees C, for lipid rotation with respect to the membrane normal. The rotational diffusion coefficient of the lipid molecules was calculated to be Dr = 2.0 x 10(9) rad2.s-1 and the apparent microviscosity in the vesicle membrane, as derived from the rotational correlation time, was eta 2 approximately 12 mPa.s. The rotational correlation time of the phosphate translocator in the membrane was only slightly dependent on the viscosity of the medium. The temperature dependence of the protein rotation also indicated that the rotation of the protein in the membrane was largely restricted and occurred mainly about the axis normal to the membrane plane. Measurements at a medium viscosity of eta 1 = 1 mPa.s yielded a value of phi r approximately 450 ns corresponding to Dr = 8.8 x 10(7) rad2.s-1 for protein rotation with respect to the membrane normal. From this value and the data of the lipid rotation, the cross-sectional area of the protein part embedded in the membrane was calculated to be approximately 9 nm2. This cross-sectional area is large enough to include at most 14 membrane-spanning helices. Our results also indicated that at lipid/protein molar ratios greater than or equal to 1.5 x 10(4): 1 aggregation occurred in the model membranes below 30 degrees C. However, above 30 degrees C and at a high dilution of the protein in the membrane it appeared that the membrane viscosity monitored by lipid and protein rotational diffusion were identical.     

Microaggregation of hormone-occupied epidermal growth factor receptors on plasma membrane preparations.

The rotational diffusion of the complexes of epidermal growth factor (EGF) with its specific receptor on plasma membrane vesicles prepared from human epidermoid carcinoma A431 cells was studied using the time-resolved polarization of phosphorescence of erythrosin-labeled hormone. The measured rotational correlation times of 16-20 microseconds at 4 degrees C are consistent with monomeric freely diffusing EGF receptor. Upon increasing the temperature to 37 degrees C, the rate of rotational diffusion slows down as evidenced by an increase in the correlation time to 75 microseconds. This finding suggests that small clusters of the occupied EGF receptor (microaggregation) form at the higher temperature, a property we have reported previously for occupied receptors on living A431 cells. Subsequent cooling of the membranes leads to a partial reversal of the microaggregation. We conclude that clustering of occupied EGF receptors can proceed at 37 degrees C in the absence of metabolic energy and external interactions, e.g. with components of the cytoskeleton, and thus reflects inherent properties of the receptor protein in its natural environment. A lag phase in the time course of microaggregation observed with the isolated membrane preparations may reflect cooperativity in the process of receptor association.     

State of aggregation of the (Ca2+ + Mg2+)-ATPase studied using saturation-transfer electron spin resonance

The state of aggregation of the (Ca2+ + Mg2+)-ATPase in the membrane of sarcoplasmic reticulum and in reconstituted membrane systems has been studied using saturation-transfer electron spin resonance (ST-ESR). Saturation-transfer ESR spectra show that in the sarcoplasmic reticulum, the ATPase is relatively free to rotate, with an effective rotational correlation time of approx. 33 microseconds at 4 degrees C, consistent with a monomeric or dimeric structure. The rate of rotation is observed to decrease with decreasing molar ratio of lipid to protein. In reconstituted systems, rotational motion of the ATPase on the millisecond time scale ceases when the lipids are in the gel phase. Addition of decavanadate, which causes the formation of crystalline arrays in negatively stained electron micrographs, results in only a small reduction in rotation rate for the ATPase in the membrane. The experiments are interpreted in terms of a short-lived (on the millisecond time scale) protein-protein interaction, with the formation of crystalline clusters of ATPase molecules which form and melt rapidly.     

Cytoskeletal restraints of band 3 rotational mobility in human erythrocyte membranes

The interaction of band 3 with cytoskeletal proteins was investigated in erythrocyte membranes by measuring the rotational mobility of band 3 using the method of transient dichroism. It was found that selective proteolysis of ankyrin, a protein known to link band 3 to the spectrin-actin network, had no significant effect on band 3 rotation. Incubating ghosts to 70 degrees C, at which temperature ankyrin is expected to be denatured, also had no effect. It thus appears probable that linkage of band 3 to the cytoskeleton via ankyrin does not act as a restraint on band 3 rotational motion. It is suggested that this is a consequence of flexibility in the cytoskeletal structure. In further investigations of the effect of heat treatment, a large enhancement of band 3 rotational mobility was found to result from incubation of intact cells for 1 h at 50 degrees C. This effect was not observed if ghosts were subjected to the same treatment, nor did it occur if the incubation of cells was performed at 47 degrees C. These findings, in combination with previous studies of band 3 rotational mobility, indicate that the interactions which restrain band 3 are likely to be more complex than commonly envisaged.     

Rotational dynamics of erythrocyte spectrin

The rotational diffusion of erythrocyte spectrin has been measured using time-resolved phosphorescence anisotropy. The anisotropy of the spectrin dimer decays to zero with a time constant of 3 microseconds at 21 degrees C. The results are compared with the correlation times predicted for the anisotropy decay of an equivalent sphere and rigid rod. The data indicate that the ribbon-like spectrin molecule possesses considerable torsional and segmental flexibility. These motions are restricted, but not abolished, when spectrin is reconstituted into cross-linked cytoskeletal protein networks, or bound to spectrin-actin depleted erythrocyte membrane vesicles.     

Influence of temperature and cholesterol on the rotational diffusion of band 3 in the human erythrocyte membrane.

Band 3 rotation in the human erythrocyte membrane is measured by observing flash-induced dichroism of eosin probes. The decay of the absorption anisotropy is found to be strongly dependent on temperature. The results are analyzed on the assumption that rotation of band 3 only occurs about the membrane normal. It is deduced that both fast and slowly rotating forms of band 3 coexist in the membrane. The equilibrium between these forms is temperature dependent, the slowly rotating species becoming increasingly dominant as the temperature is reduced. Plots of the fractional distribution of the different species against temperature show a marked change of slope at around 37--40 degrees C. The effects are essentially reversible over the range 1--45 degrees C and independent of the presence of the spectrin--actin network. The results could be due to temperature-dependent protein--protein associations mediated either by a protein conformational change or by lipid phase segregation. In further experiments, the cholesterol content of the erythrocyte membrane is varied by incubation with lipid vesicles. No significant changes in the rotational diffusion of band 3 are observed following variation of membrane cholesterol/phospholipid mole ratios over the range 0.34--1.66. This is a surprising result in view of the well-known effects of cholesterol on lipid fluidity.     

A spin-label and hydrogen-deuterium exchange reaction kinetics study of protein-lipid interactions in lipid-replaced Ca2+-ATPase of rabbit skeletal muscle sarcoplasmic reticulum

The Ca2+-ATPase of sarcoplasmic reticulum from rabbit skeletal muscle was incorporated into vesicles made from dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine. The Ca2+-ATPase activity of these reconstituted membranes became appreciable above 20 degrees C and 30 degrees C, respectively, in accord with the results of previous investigators. Measurement by the spin-labeling technique of the fluidity of the bulk lipid revealed the gel-to-liquid crystalline phase transition at 29 degrees C and 39 degrees C, respectively, while the fluidity of the boundary lipid in both samples was found to be low throughout the temperature range studied. The rotational mobility of the Ca2+-ATPase protein in both samples, measured by saturation transfer electron spin resonance, was also very low throughout the temperature range studied and its temperature-dependence did not show any break or jump corresponding to the phase transition of the bulk lipid. On the other hand, the structural fluctuation of the Ca2+-ATPase protein in dimyristoylphosphatidylcholine-recombinant, measured in terms of hydrogen-deuterium exchange reaction kinetics, showed a jump at about 27 degrees C, apparently in accordance with the phase transition of the bulk lipid. Results obtained in this study suggested that the Ca2+-ATPase protein molecules are in an aggregated state in these reconstituted membranes and that the Ca2+-ATPase activity is neither directly correlated to the fluidity of the boundary lipid nor to the rotational mobility of the Ca2+-ATPase, contrary to the suggestions of previous investigators (Hesketh et al. (1976) Biochemistry 15, 4145-4151; Hidalgo et al. (1978) J. Biol. Chem. 253, 6879-6887).     

Spectrin-dependent and -independent association of F-actin with the erythrocyte membrane

Binding of F-actin to spectrin-actin-depleted erythrocyte membrane inside-out vesicles was measured using [3H]F-actin. F-actin binding to vesicles at 25 degrees C was stimulated 5-10 fold by addition of spectrin dimers or tetramers to vesicles. Spectrin tetramer was twice as effective as dimer in stimulating actin binding, but neither tetramer nor dimer stimulated binding at 4 degrees C. The addition of purified erythrocyte membrane protein band 4.1 to spectrin- reconstituted vesicles doubled their actin-binding capacity. Trypsinization of unreconstituted vesicles that contain < 10% of the spectrin but nearly all of the band 4.1, relative to ghosts, decreased their F-actin-binding capacity by 70%. Whereas little or none of the residual spectrin was affected by trypsinization, band 4.1 was significantly degraded. Our results show that spectrin can anchor actin filaments to the cytoplasmic surface of erythrocyte membranes and suggest that band 4.1 may be importantly involved in the association.     

Rotational motion of yeast cytochrome oxidase in phosphatidylcholine complexes studied by saturation-transfer electron spin resonance

Cytochrome oxidase from yeast has been covalently labeled with a nitroxide derivative of maleimide and reconstituted in lipid-substituted complexes with dimyristoyl-, dioleoyl-, or dielaidoyl-phosphatidylcholine. The rotational mobility of the enzyme in the complexes has been studied as a function of temperature and time, and of lipid/protein ratio, using saturation-transfer electron spin resonance spectroscopy. For complexes with dimyristoylphosphatidylcholine, the rotational mobility of the protein decreases abruptly below the gel-to-fluid-phase transition. This change is accompanied by a lateral segregation of the protein, as seen by freeze-fracture electron microscopy, and by an increase in the activation energy for the enzymatic activity. A time-dependent decrease in the rotational motion of the protein is observed on incubating at temperatures in the fluid phase of the lipid. This corresponds with a time-dependent loss of enzyme activity observed on incubation at temperatures in the fluid phase, but not at temperatures in the gel phase, over a period of 3 h. The rotational mobility decreases with increasing protein concentration in the complexes, both in the fluid and in the gel phases. The dependence of the protein mobility on lipid/protein ratio can be interpreted quantitatively in terms of the effect of increased random protein-protein contacts in the fluid phase. The maximum limiting rotational correlation time for the protein diffusion at high lipid/protein ratios in the fluid phase is tau R[[ approximately equal to 25 microseconds, suggesting that the protein is present as either a monomer or more probably a dimer in the reconstituted membrane.     

Characterization of interaction between Tb3+ and porcine intestinal brush-border membranes

Effects of ionic strength and temperature on the interaction between Tb3+ and porcine intestinal brush-border membrane vesicles were studied. When Tb3+ was added to the vesicle suspension, Tb3+ fluorescence increased with increasing concentration of Tb3+, showing a saturation. The apparent dissociation constant of one of at least two components of this binding reaction was estimated to be about 12.5 microM at 25 degrees C, pH 7.4. But the affinity of Tb3+ for the membrane vesicles was variable with changes of ionic strength and temperature. The affinity was lowered by addition of KCl to medium and by increase of temperature above 30 degrees C. In addition, temperature-induced change in the affinity of Tb3+ for the membranes was reversible over a temperature range from 13 to 46 degrees C. Temperature-dependence profiles of the excimer formation efficiency of pyrene-labeled membranes and of the harmonic mean of the rotational relaxation times of pyrene molecules in the membranes revealed that the phase transition of the membrane lipids occurs at about 30 degrees C. Based on these results, characteristics of Tb3+ binding to the membranes are discussed in relation to the nature of lipid phase and surface charges of the membranes.     

Electron spin resonance study of phospholipid membranes employing a comprehensive line-shape model

The electron spin resonance spectra of the 1-myristoyl-2-[6-(4,4-dimethyloxazolidine-N-oxyl)myristoyl]-sn-glycero- 3-phosphocholine spin-label in highly oriented, fully hydrated bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine have been studied as a function of temperature and magnetic field orientation. The oriented spectra show clear indications of slow motional components (rotational correlation times greater than 3 ns) even in the fluid phase (T greater than 23 degrees C), indicating that motional narrowing theory is not applicable to the spectral analysis. The spectra have been simulated by a comprehensive line-shape model that incorporates trans-gauche isomerization in addition to restricted anisotropic motion of the lipid long molecular axis and that is valid in all motional regimes. In the gel (L beta') phase the spin-label chains are found to be tilted at 28 degrees with respect to the normal of the orienting plane. In the intermediate (P beta') phase there is a continuous distribution of tilt angles between 0 degrees and 25 degrees. In fluid (L alpha) phase there is no net tilt of the lipid chains. The chains rotate at an intermediate rate about their long axis in the fluid phase (tau R,parallel = 1.4-6.6 ns for T = 50-25 degrees C), but the reorientation of the chain axis is much slower (tau R, perpendicular= 13-61 ns for T = 50-25 degrees C), whereas trans-gauche isomerization (at the C-6 position) is rapid (tau J less than or equal to 0.2 ns). Below the chain melting transition both chain reorientation and chain rotation are at the ESR rigid limit (tau R greater than or equal to 100 ns), and trans-gauche isomerization is in the slow-motion regime (tau J = 3.7-9.5 ns for T = 22-2 degrees C). The chain order parameter increases continuously with decreasing temperature in the fluid phase (SZZ = 0.47-0.61 for T = 50-25 degrees C), increases abruptly on going below the chain melting transition, and then increases continuously in the intermediate phase (SZZ = 0.79-0.85 for T = 22-14 degrees C) to an approximately constant value in the gel phase (SZZ congruent to 0.86 for T = 10-2 degrees C).(ABSTRACT TRUNCATED AT 400 WORDS)     

Rotational motion and flexibility of Ca2+,Mg2+-dependent adenosine 5'-triphosphatase in sarcoplasmic reticulum membranes

Ca2+,Mg2+-dependent adenosine 5'-triphosphatase (ATPase) in sarcoplasmic reticulum vesicles is labeled with the triplet probe, 5-iodoacetamidoesin. Rotational mobility of the ATPase is investigated by measuring flash-induced transient dichroism of the eosin probe. The absorption anisotropy measured 20 mus after the exciting flash is found to be small at 37 degrees C but increases considerably with decreasing temperature and upon fixation with glutaraldehyde. A purified Ca2+,Mg2+-dependent ATPase preparation partially depleted of membrane lipids exhibits similar properties. The low value of the anisotropy at 37 degrees C is due to the existence of a fast motion which in part is assigned to independent segmental motion of the protein. This internal flexibility of the ATPase may have considerable significance for the functional properties of the enzyme. At times longer than 20 mus, the anisotropy decays with a time constant which varies from approximately 90 mus at 0 degrees C to approximately 40 mus at 37 degrees C. This decay is assigned to rotation of the ATPase about an axis normal to the plane of the membrane. There is some evidence for self-aggregation of the protein at lower temperatures.     

The use of the fluorescent probe alpha-parinaric acid to determine the physical state of the intracytoplasmic membranes of the photosynthetic bacterium, Rhodopseudomonas sphaeroides.

alpha-Parinaric acid has been used to determine the degree of ordering of the hydrocarbon region of purified intracytoplasmic membranes of Rhodopseudomonas sphaeroides. The usefulness of alpha-parinaric acid as a probe of membrane fluidity was established by comparison of its fluorescent properties in phosphatidylcholine vesicles with those of the more commonly used fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene. Both fluorescent probes were shown to monitor similar environments in the phosphatidylcholine vesicles when the phospholipids were maintained at temperatures above their phase transition temperature. The rotational mobility of alpha-parinaric acid in the intracytoplasmic membranes was determined from 0 to 50 degrees C, a region where no phase transitions were detectable. The rotational mobility of alpha-parinaric acid dissolved in vesicles formed from total extracted intracytoplasmic membrane phospholipids, was 2--3-fold greater than that measured in the intact intracytoplasmic membranes; demonstrating that the presence of protein greatly reduces the mobility of the phospholipid acyl chains of the intracytoplasmic membranes. Due to the high protein content of these membranes, the perturbing effect of protein on acyl chain mobility may extend to virtually all the intracytoplasmic membrane phospholipid.     

Loss of tRNA 5-methyluridine methyltransferase and pseudouridine synthetase activities in 5-fluorouracil and 1-(tetrahydro-2-furanyl)-5-fluorouracil (ftorafur)-treated Escherichia coli

The degradation of human erythrocyte membrane proteins in relation to the identification of the monosaccharide transporter has been investigated in whole membrane preparations and membrane protein extracts by polyacrylamide gel electrophoresis in sodium n-dodecyl sulphate and iodine-125 labelling. Evidence is presented for the degradation of band 3 polypeptide to lower molecular weight material some of which appears in region 4.5 of the polyacrylamide gel electrophoresis profile. It is found that the degradation process is inhibited by phenylmethylsulphonyl fluoride and is only significant in membrane extracts in the absence of detergent (Triton X-100) and on prolonged incubation at 37 degrees C, conditions which do not prevail during the isolation of membrane protein extracts for reconstitution studies. Extracts of band 3 and band 4.5 have been prepared and reconstituted in bilayer lipid membranes. The permeabilities of the reconstituted systems to D-glucose have been investigated and it is found that only bilayers incorporating band 4.5 exhibited enhanced monosaccharide transport. A linear relationship between D-glucose transport and the concentration of protein in the aqueous phase bathing the bilayers suggests a partitioning of the protein into the bilayer. Reconstitution is stereospecific and inhibited by cytochalasin B.     

Effects of melittin on molecular dynamics and Ca-ATPase activity in sarcoplasmic reticulum membranes: time-resolved optical anisotropy.

We have studied the effect of melittin, a basic membrane-binding peptide, on Ca-ATPase activity and on protein and lipid dynamics in skeletal sarcoplasmic reticulum (SR), using time-resolved phosphorescence and fluorescence spectroscopy. Melittin completely inhibits Ca-ATPase activity, with half-maximal inhibition at 9 +/- 1 mol of melittin bound to the membrane per mole of ATPase (0.1 mol of melittin per mole of lipid). The time-resolved phosphorescence anisotropy (TPA) decay of the Ca-ATPase labeled with erythrosin isothiocyanate (ERITC) shows that melittin restricts microsecond protein rotational motion. At 25 degrees C in the absence of melittin, the TPA is characterized by three decay components, corresponding to a rapid segmental motion (correlation time phi 1 = 2-3 microseconds), the uniaxial rotation of monomers or dimers (phi 2 = 16-22 microseconds), and the uniaxial rotation of larger oligomers (phi 3 = 90-140 microseconds). The effect of melittin is primarily to decrease the fraction of the more mobile monomer/dimer species (A2) while increasing the fractions of the larger oligomer (A3) and very large aggregates (A infinity). Time-resolved fluorescence anisotropy of the lipid-soluble probe diphenylhexatriene (DPH) shows only a slight increase in the lipid hydrocarbon chain effective order parameter, corresponding to an increase in lipid viscosity that is too small to account for the large decrease in protein mobility or inhibition of Ca-ATPase activity. Thus the inhibitory effect of melittin correlates with its capacity to aggregate the Ca-ATPase and is consistent with previously reported inhibition of this enzyme under conditions that increase protein-protein interactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamic structures of adrenocortical cytochrome P-450 in proteoliposomes and microsomes: protein rotation study.

Purified adrenocortical microsomal cytochromes P-45017 alpha,lyase and P-450C21 were reconstituted with and without NADPH-cytochrome P-450 reductase in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles at a lipid to P-450 ratio of 35 (w/w) by cholate dialysis procedures. Trypsinolysis revealed that a considerable part of each P-450 molecule is deeply embedded in the lipid bilayer, on the basis of the observation of no detectable digestion for P-45017 alpha,lyase and the proteolysis-resistant membrane-bound heavy fragments for P-450C21. Rotational diffusion was measured in proteoliposomes and adrenocortical microsomes by observing the decay of absorption anisotropy, r(t), after photolysis of the heme-CO complex. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The absorption anisotropy decayed within 1-2 ms to a time-independent value r3. Coexistence of a mobile population with an average rotational relaxation time phi of 138-577 microseconds and immobile (phi > or = 20 ms) populations of cytochrome P-450 was observed in both phospholipid vesicles and microsomes. Different tilt angles of the heme plane from the membrane plane were determined in proteoliposomes to be either 47 degrees or 63 degrees for P-45017 alpha,lyase from [r3/r(0)]min = 0.04 and either 38 degrees or 78 degrees for P-450C21 from [r3/r(0)]min = 0.19, when these P-450s were completely mobilized by incubation with 730 mM NaCl. Very different interactions with the reductase have been observed for the two P-450s in proteoliposomes. In the presence of the reductase, the mobile population of cytochrome P-450C21 was increased significantly from 79% to 96% due to dissociation of P-450 oligomers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rotational dynamics of the Ca-ATPase in sarcoplasmic reticulum studied by time-resolved phosphorescence anisotropy

We have investigated the microsecond rotational motions of the Ca-ATPase in rabbit skeletal sarcoplasmic reticulum (SR), by measuring the time-resolved phosphorescence anisotropy of erythrosin 5-isothiocyanate (ERITC) covalently and specifically attached to the enzyme. Over a wide range of solvent conditions and temperatures, the phosphorescence anisotropy decay was best fit by a sum of three exponentials plus a constant term. At 4 degrees C, the rotational correlation times were phi 1 = 13 +/- 3 microseconds, phi 2 = 77 +/- 11 microseconds, and phi 3 = 314 +/- 23 microseconds. Increasing the solution viscosity with glycerol caused very little effect on the correlation times, while decreasing the lipid viscosity with diethyl ether decreased the correlation times substantially, indicating that the decay corresponds to rotation of the protein within the membrane, not to vesicle tumbling. The normalized residual anisotropy (A infinity) is insensitive to viscosity and temperature changes, supporting the model of uniaxial rotation of the protein about the membrane normal. The value of A infinity (0.20 +/- .02) indicates that each of the three decay components can be analyzed as a separate rotational species, with the preexponential factor Ai equal to 1.25X the mole fraction. An empirically accurate measurement of the membrane lipid viscosity was obtained, permitting a theoretical analysis of the correlation times in terms of the sizes of the rotating species. At 4 degrees C, the dominant correlation time (phi 3) is too large for a Ca-ATPase monomer, strongly suggesting that the enzyme is primarily aggregated (oligomeric).(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR structural analysis of a membrane protein: bacteriorhodopsin peptide backbone orientation and motion

In reconstituted vesicles above the lipid phase transition temperature, bacteriorhodopsin (BR) undergoes rotational diffusion about an axis perpendicular to the plane of the bilayer [Cherry, R. J., Muller, U., & Schneider, G. (1977) FEBS Lett. 80, 465]. This diffusion narrows the 13C NMR powder line shape of the BR peptide carbonyls. In contrast, BR in native purple membrane is relatively immobile and exhibits a rigid-lattice powder line shape. By use of the principal values of the rigid-lattice chemical shift tensor and the motionally narrowed line shape from the reconstituted system, the range of Euler angles of the leucine peptide groups relative to the diffusion axis has been calculated. The experimentally observed line shape is inconsistent with those expected for structures which consist entirely of either alpha helix or beta sheet perpendicular to the membrane or beta sheet tilted at angles up to about 60 degrees from the membrane normal. However, for two more complex structural models, the predicted line shapes agree well with the experimental one. These are, first, a structure consisting entirely of alpha1 helices tilted at 20 degrees from the membrane normal and, second, a combination of 60% alpha II helix perpendicular to the membrane plane and 40% antiparallel beta sheet tilted at 10-20 degrees from the membrane normal. The results also indicate that the peptide backbone of bacteriorhodopsin in native purple membrane is extremely rigid even at 40 degrees. The experiments presented here demonstrate a new approach, using solid-state nuclear magnetic resonance (NMR) methods, for structural studies of transmembrane proteins in fluid membrane environments, either natural or reconstituted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Competitive inhibition of the inverting beta-xylosidase of Bacillus pumilus 12 by monosaccharide derivatives of different structural and conformational types. A possible natural substrate.

We studied the rotational mobility of the Ca2+ + Mg2+-activated ATPase in skeletal-muscle sarcoplasmic-reticulum vesicles, using time-resolved measurements of the depolarization of laser-flash-excited phosphorescence of the extrinsic triplet probe erythrosin. Our results are in general agreement with those of others [Bürkli & Cherry (1981) Biochemistry 20, 138-145] obtained by linear dichroism methods. In addition, we directly observed fast depolarization in the 1-5 microseconds time range that can be attributed to limited motion of part of the protein (segmental motion). Temperature-dependent changes in phosphorescence anisotropy indicated the onset of a conformational change in structure of the Ca2+ + Mg2+-activated ATPase at 11-13 degrees C. We also describe the synthesis of 5-iodoacetamidoerythrosin.     

Membrane fusion activity of reconstituted vesicles of influenza virus hemagglutinin glycoproteins

Reconstituted vesicles of hemagglutinin glycoproteins into egg yolk phosphatidylcholine/spin-labeled phosphatidylcholine/cholesterol (molar ratio 1.6:0.4:1) were prepared by dialysis. Preparations at appropriate protein-to-lipid ratios (1:44 and 1:105 mol/mol) contained vesicles with a diameter of 100-300 nm and a high density of spikes on the surface. These vesicles showed low pH-induced membrane fusion activity. At pH 5.2 and 37 degrees C, fusion with erythrocyte membranes took place very rapidly within 1-2 min and reached a plateau at 63-66% fusion. The fusion was negligibly small at neutral pH and was induced to occur at pH values lower than 6.0. The reconstituted vesicles caused hemolysis and fusion of human erythrocyte cells in the same pH range as that of the fusion with erythrocyte membranes. The low pH-induced fusion activity of the reconstituted vesicles is essentially the same as that of the parent virus. These vesicles can be used to deliver some reagents or drugs into target cell cytoplasm via fusion at lysosomes.