RESUMEN

The application of two-dimensional Fourier-transform electron-spin-resonance (2D-FT-ESR) to the study of lipid/gramicidin A (GA) interactions is reported. It is shown that 2D-FT-ESR spectra provide substantially enhanced spectral resolution to changes in the dynamics and ordering of the bulk lipids (as compared with cw-ESR spectra), that result from addition of GA to membrane vesicles of dipalmitoylphosphatidylcholine (DPPC) in excess water containing 16-PC as the lipid spin label. The agreement between the theory of Lee, Budil, and Freed and experimental results is very good in the liquid crystalline phase. Both the rotational and translational diffusion rates of the bulk lipid are substantially decreased by addition of GA, whereas the ordering is only slightly increased, for a 1:5 ratio of GA to lipid. The slowing effect on the diffusive rates of adding GA in the gel phase is less pronounced. It is suggested that the spectral fits in this phase would be improved with a more detailed dynamic model. No significant evidence is found in the 2D-FT-ESR spectra for a second immobilized component upon addition of GA, which is in contrast to cw-ESR. It is shown from simulations of the observed 2D-FT-ESR spectra that the additional component seen in cw-ESR spectra, and usually attributed to "immobilized" lipid, is inconsistent with its being characterized by increased ordering, according to a model proposed by Ge and Freed, but it would be consistent with the more conventional model of a significantly reduced diffusional rate. This is because the 2D-FT-ESR spectra exhibit a selectivity, favoring components with longer homogeneous relaxation times, T2. The homogeneous linewidths of the 2D-FT-ESR autopeaks appear to broaden as a function of mixing time. This apparent broadening is very likely due to the process of cooperative order director fluctuations (ODF) of the lipids in the vesicle. This real-time observation of ODF is distinct from, but appears in reasonable agreement with, NMR results. It is found that addition of GA to give the 1:5 ratio has only a small effect on the ODF, but there is a significant temperature dependence.

Asunto(s)

RESUMEN

A two-dimensional Fourier Transform ESR (2D FT ESR) spectrometer operating at 9.25 and 17.35 GHz is described. The Ku-band bridge uses an efficient heterodyne technique wherein 9.25 GHz is the intermediate frequency. At Ku-band the sensitivity is increased by almost an order of magnitude. One may routinely collect a full 2D ELDOR spectrum in less than 20 min for a sample containing 0.5-5 nmol of nitroxide spin-probe in the slow-motional regime. Broad spectral coverage at Ku-band is obtained by use of a bridged loop-gap resonator (BLGR) and of a dielectric ring resonator (DR). It is shown that an even more uniform spectral excitation is obtained by using shorter microwave pulses of about 3 ns duration. The dead-time at Ku-band is just 30-40 ns, yielding an improved SNR in 2D ELDOR spectra of nitroxide spin-probes with T2 as short as 20-30 ns. A comparison of 2D ELDOR spectra obtained at 9.25 and 17. 35 GHz for spin-labeled phospholipid probes (16PC) in 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) membrane vesicles showed that both spectra could be satisfactorily simulated using the same set of model parameters even though they are markedly different in appearance. The improved sensitivity and shorter dead-time at Ku-band made it possible to obtain orientation-dependent 2D ELDOR spectra of the Cholestane (CSL) spin-probe in macroscopically aligned lipid bilayers of egg yolk PC using samples containing only 1 mg of lipid and just 5 nmol of spin-probe.

Asunto(s)

RESUMEN

The first two-dimensional Fourier-transform electron spin resonance (2D-FT-ESR) studies of nitroxide-labeled lipids in membrane vesicles are reported. The considerable enhancement this experiment provides for extracting rotational and translational diffusion rates, as well as orientational ordering parameters by means of ESR spectroscopy, is demonstrated. The 2D spectral analysis is achieved using theoretical simulations that are fit to experiments by an efficient and automated nonlinear least squares approach. These methods are applied to dispersions of 1-palmitoyl-2oleoyl-sn-glycerophosphatidylcholine (POPC) model membranes utilizing spin labels 1-palmitoyl-2-(16-doxyl stearoyl) phosphatidylcholine and the 3-doxyl derivative of cholestan-3-one (CSL). Generally favorable agreement is obtained between the results obtained by 2D-FT-ESR on vesicles with the previous results on similar systems studied by continuous wave (cw) ESR on aligned samples. The precision in determining the dynamic and ordering parameters is significantly better for 2D-FT-ESR, even though the cw ESR spectra from membrane vesicles are resolved more poorly than those from well aligned samples. Some small differences in results by the two methods are discussed in terms of limitations of the methods and/or theoretical models, as well as possible differences between dynamic molecular structure in vesicles versus aligned membranes. An interesting observation with CSL/POPC, that the apparent homogeneous linewidths seem to increase in "real time," is tentatively attributed to the effects of slow director fluctuations in the membrane vesicles.

Asunto(s)

RESUMEN

Sickle cell anemia results from the formation of hemoglobin S fibers in erythrocytes, and a greater understanding of the structure of these fibers should provide insights into the basis of the disease and aid in the development of effective antisickling agents. Improved reconstructions from electron micrographs of negatively stained single hemoglobin S fibers or embedded fiber bundles reveal that the 14 strands of the fiber are organized into pairs. The strands in each of the seven pairs are half-staggered, and from longitudinal views the polarity of each pair can be determined. The positions of the pairs and their polarities (three in one orientation; four in the opposite orientation) suggest a close relationship with the crystals of deoxyhemoglobin S composed of antiparallel pairs of half-staggered strands.

Asunto(s)

RESUMEN

The innermost chorionic layer (ICL) within egg shells of Drosophila is composed of a family of related, thin three-dimensional crystals that form a continuous sheath encapsulating the egg shell lumen. Junctions formed by interdigitating lattices play a central role in the construction of this macroscopic assembly. The three-dimensional structure of a two-dimensional crystal isolated from the ICL, with a primitive lattice angle delta of 90 degrees, has been determined from a complete tilt series of a negatively contrasted specimen at a resolution of 25 A. Inspection of the three-dimensional transform after data merging revealed that the space group is c222 and this symmetry was employed to generate a three-dimensional structure. The basic structural unit of the ICL is an octamer, described formally as a tetramer of dimers with point group symmetry 222. There are two classes of dimer in the octamer designated alpha and beta. The chorin octamer is composed of two classes of bent dimers, which make intramolecular contacts at the top and bottom of the molecule. The alpha-dimers are curved outwards away from the crystallographic 2-fold axis, while the beta-dimers are curved towards the molecular center. In addition, lattice contacts are formed primarily by beta-chorin dimers at both the top and bottom surfaces of the unit cell. The molecular weight of a chorin octamer determined from the analysis is about 6 X 10(5). The conformation of the chorin octamer determined here suggests that permutations of a basic molecular mechanism may be adequate to explain both the observed lattice polymorphisms of the ICL and the formation of interplate junctions necessary for the assembly of the macroscopic sheath.

Asunto(s)

RESUMEN

Images of negatively stained fibers of sickle cell hemoglobin have been analyzed by cross-correlation methods. These methods are used to compensate for the curvature and variable repeat distance characteristic of negatively stained fibers. Averaged images obtained by the correlation procedure display considerably more detail than the filtered images obtained earlier by Fourier methods. The averaged images are sufficiently detailed that the back-projection method for obtaining cross-sections can now be applied directly to the correlation-averaged images. This method was used previously to deduce the 14-strand structure on Fourier-filtered images that incorporated only the near-equatorial maxima. In this way the 14-strand structure has been reconfirmed without utilizing the partial Fourier reconstructions employed earlier that might conceivably have introduced spurious features. In addition, application of the correlation procedure with and without inversion of the reference reveals a consistent polarity in all of the fibers examined. The confirmation of the 14-strand structure by a new procedure and the determination of fiber polarity would appear to eliminate the alternative model of the fibers with a 16-strand structure with equal numbers of strands (eight) of each polarity.

Asunto(s)

RESUMEN

Zinc-induced tubulin sheets without microtubule-associated proteins (MAPs) were assembled from tubulin purified by phosphocellulose chromatography. Large, open sheets were obtained in five-minute incubations at pH 5.7. Electron micrographs of negatively stained sheets showed a protofilament arrangement similar to that observed for zinc-induced sheets with MAPs but with altered lattice parameters. The spacings measured from optical diffraction patterns demonstrated that the protofilaments were 2.2 A closer together in the sheets without MAPs. Each MAP-free sheet was also divided roughly in half by a discontinuity which was parallel to the protofilaments and the relationship between the two domains was deduced from computed transforms. Two-dimensional image processing was carried out by conventional Fourier techniques and by correlation analysis. The correlation analysis improved the reconstructions in this application, with the resolution limited by the inherent properties of the negative stain method to about 14 A. A prominent feature of the computed reconstructions was an alternation of light and dark protofilaments due to differential staining, as revealed by a study of folded sheets. Neighboring protofilaments are related by a 2-fold screw axis, as they are in zinc-induced sheets with MAPs, but the symmetry is masked by the differential staining. The major effect of MAP removal on the structure of the sheets is that the bilobed structure of alternate tubulin subunits is no longer observed. This observation and the closer spacing of protofilaments is consistent with the postulate that some of the MAP molecules lie in the groove between protofilaments and bind to several tubulin dimers.

Asunto(s)

RESUMEN

The morphology of F1-ATPases lacking one or more small subunits has been investigated by minimal-beam electron microscopy of close-packed monolayers of molecules. Computer-based rotational analyses of single molecules were performed on reconstituted 3-subunit F1-ATPase (-delta epsilon) from Escherichia coli and both 3-subunit (-delta epsilon) and 4-subunit (-delta) F1-ATPase from chloroplasts. Optical diffraction measurements of close-packed arrays revealed maximal dimensions of 122 +/- 4 A and 129 +/- 9 A for 3-subunit ECF1 and 4-subunit CF1, respectively. Molecules which displayed either hollow or solid hexagonal morphologies were observed in all preparations. Averaged reconstructions were obtained from molecules with hollow morphologies in 3-subunit preparations and demonstrated strong hexagonal symmetry in projection with a central, stain-filled cavity. The average reconstruction obtained from molecules with the solid morphology in 4-subunit CF1 preparations, was also strongly hexagonal with six peripheral units ringed about a central subunit. Differences between hollow and solid morphologies cannot be attributed solely to the presence or absence of the delta and epsilon subunits; therefore, the two image types may represent staining variants of a common structure. Overall, the reconstructions are consistent with an alpha 3 beta 3 gamma stoichiometry for the coupling factors from both E. coli and chloroplasts.

Asunto(s)

RESUMEN

Hybrid hemoglobin molecules prepared with beta chains from hemoglobin S (beta 6 Glu leads to Val) and alpha chains from hemoglobin Sealy (alpha 47 Asp leads to His) form fibers with a novel structure. In contrast to the typical fibers of hemoglobin S with an average diameter of 22 nm and a solid cross section composed of 10 outer filaments surrounding a 4-filament core, the fibers of the alpha Sealy2 beta S2 hybrid are much larger, with a mean diameter of 32 nm and a unique double-hollow arrangement of filaments. Sealy--S fibers can be described by a model in which the two pairs of filaments most readily lost from fibers of hemoglobin S are missing to form the hollow regions, with an additional sheath of filaments added to form the overall larger structure.

Asunto(s)

RESUMEN

Large negatively-stained two-dimensional tubulin arrays induced by incubation with zinc were examined using electron microscopy. When Fourier methods are applied for the reconstruction, the resolution depends critically on the order of these arrays. The real space cross-correlation method as described in this paper, however, can be satisfactorily applied to disordered arrays. The method involves selection of a small area which is used as a reference to locate correlation peaks in other regions. In this way the locations of various unit cells are determined, often at points which deviate from the exact lattice. The unit cells at their determined locations are averaged, and the average is used as an improved reference in another search for correlation peaks. The process can be iterated to refine the reference and improve the correlation mapping. If necessary, poorly correlating unit cells can be ignored in the averaging, and rotational as well as translational corrections can be considered; but neither of these steps were found to be necessary in the studies on the zinc-induced tubulin sheets reported here. When both correlation and Fourier methods were compared for the same areas of selected sheets, the two methods gave comparable results for relatively well ordered regions, but the correlation images were superior for the more poorly ordered regions. In addition, even for the relatively well ordered regions the correlation images revealed some details that were lacking in the simple Fourier reconstructions but did appear in more extensive studies involving three-dimensional reconstruction. A direct comparison of the efficacy of real space and Fourier space reconstruction methods is presented using disordered model data.

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)

RESUMEN

Fibers of deoxyHb S have been investigated by thin-section electron microscopy, utilizing a tannic acid embedding procedure. On the basis of numerous measurements of cross-sectional center-to-center distances for adjacent fibers in pairs or arrays, fiber diameters (mean +/- SD) of 205 +/- 5 A in embedded cells and 212 +/- 8 A in embedded hemolysates were obtained. This is an agreement with values obtained by conventional embedding procedures [Crepeau, R. H., Dykes, G., Garrell, R. L. & Edelstein, S. J. (1978) Nature (London) 274, 616--617]. The use of tannic acid has resulted in improved resolution of fiber cross sections, revealing individual strands of Hb S molecules. Because the section thickness corresponds to approximately one-fifth of the fiber helical repeat distance, the strands in projection superimpose to form characteristic image patterns. Additional superposition patterns arise in sections taken at small deviations from perpendicularity to the longitudinal fiber axis. These patterns are consistent with the 14-strand structure for hemoglobin S fibers [Dykes, G., Crepeau, R. H. & Edelstein, S. J. (1978) Nature (London) 272, 506--510], as indicated by computer models of cross-sectional patterns for various thicknesses and angular deviations of sections.

Asunto(s)

RESUMEN

Electron microscopic techniques have been used to reveal two classes of subunits of tubulin in ordered arrays. Presumably the two classes correspond to the alpha and beta polypeptide chains of tubulin that have been distinguished by chemical criteria. The two types of subunits alternate along individual protofilaments in microtubules, microtubule-precursor sheets, and extended zinc-tubulin sheets. The resolution of the two types of polypeptide chains is achieved by improved negative staining methods which produce micrographs with layer lines at 28 A(-1) and 84 A(-1) in optical or computed transforms, in addition to the layer lines at 21 A(-1) and 42 A(-1) described previously [Crepeau, R. H., McEwen, B., Dykes, G. & Edelstein, S. J. (1977) J Mol. Biol. 116, 301-315]. In microtubules or microtubule-precursor sheets, adjacent protofilaments are staggered by about 10 A, but parallel, in the sense that the alpha-beta vector points in the same direction for all of the protofilaments of the microtubule. However, for the sheets assembled in the presence of zinc, adjacent protofilaments are staggered by about 21 A and oriented in an antiparallel arrangement with alternate protofilaments related by a 2-fold screw axis. The antiparallel alignment of the protofilaments in the zinc-tubulin sheets accounts for their planarity (no tubular structures are found in the presence of moderate concentrations of zinc), since the intrinsic curvature found with parallel alignment of protofilaments in the absence of zinc would be cancelled by the antiparallel arrangement.

Asunto(s)

Asunto(s)

RESUMEN

Three-dimensional reconstruction of electron micrographs of the 20-nm diameter fibres of HbS reveals an inner helical core of four strands surrounded by an outer helix of 10 strands to give a total of 14 strands. The strands are arranged with roughly hexagonal packing to produce an unusual helical structure which features a variety of intermolecular contacts and a non-circular cross section.

Asunto(s)

Asunto(s)

Asunto(s)