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1.
Light meromyosin paracrystals have been studied by electron microscopy combined with optical diffraction in order to understand how the tails of the myosin molecules might pack in the backbone of muscle thick filaments. The forms of paracrystal investigated were all spindle-shaped structures with an axial periodicity of either 43 nm or 14.3 nm or hybrids involving aspects of both repeats. Transverse sections show that they are not smooth but polygonal in outline. Analysis of the band patterns in negatively stained specimens indicates that the molecular arrangement in the paracrystals involves both parallel and antiparallel interactions. A parallel axial displacement of the molecules by 43 nm is intrinsic to all forms of paracrystal investigated. The principal antiparallel overlap between molecules appears to be one of 84 nm, and it is suggested that an antiparallel dimer is the structural unit in the paracrystals. The role of the interactions leading to these displacements in the formation of the thick filament backbone is discussed.  相似文献   

2.
The molecular packing of magnesium paracrystals of α-tropomyosin has been examined by electron microscopy. Previous work (Caspar et al., 1969) had shown that these structures are composed of antiparallel arrays of molecules and we have now studied the relative positions of the molecules by matching the banding patterns of paracrystals positively stained with uranyl acetate to the sequence of the molecule. The overlap between the C-termini of the molecules in the unit cell is 175 ± 2 residues and the overlap of the N-termini lies in the range 107 to 122 residues. In the long overlap region (between C-termini), and probably also in the short overlap region, the molecular packing is such that the periodic zones of negative charge present in the sequence (Stewart & McLachlan, 1975) lie opposite one another. We propose that magnesium bridges between opposing negative charges contribute strongly to the stability of the structure. We confirm earlier work (Stewart, 1975b) on the absolute orientation of the molecules in the paracrystal: the troponin binding site on tropomyosin is approximately 130 Å from the C-terminus, and Cys190 is within 10 to 15 Å units of the C-C dyad.  相似文献   

3.
Electron micrographs of magnesium paracrystals of α-tropomyosin stained with uranyl acetate show a repeating pattern of 14 dark bands. Previous studies (Caspar et al., 1969; Ohtsuki, 1974) have shown that the molecules in the paracrystal lie antiparallel with their ends near two prominent white bands. These white lines divide the pattern into two zones containing nine and five dark bands, respectively, with the longer zone corresponding to the overlap between C termini. The present study shows that the intensity of the prominent white lines is reduced after digesting tropomyosin with carboxypeptidase A. This implies that, even in supposedly positively stained material, the white lines result from the exclusion of residual negative stain by the local thickening associated with the overlap of the ends of consecutive parallel molecules (NC overlap). Computer image processing and least-squares analysis have been employed to relate the positively stained patterns observed in both digested and undigested material to molecular positions and the amino acid sequence. Over a range of different staining criteria, it is shown that the pattern is fitted best when the C termini overlap by 176 ± 5 residues and the ends of consecutive parallel molecules overlap by 11 ± 5 residues. Uranyl ions appear to bind to carboxyl groups in the structure unless they form salt bridges with basic residues or they lie in the innermost positions of the tropomyosin coiled coil. Systematic differences between predicted and observed patterns near the molecular ends suggest that the conformation of the NC overlap may not be completely α-helical. A model with a globular N terminus and an extended C terminus is more consistent with the observed staining patterns and also offers an explanation for some other observations.  相似文献   

4.
Microtubule (MT) nucleation not only occurs from centrosomes, but also in large part from dispersed nucleation sites. The subsequent sorting of short MTs into networks like the mitotic spindle requires molecular motors that laterally slide overlapping MTs and bundling proteins that statically connect MTs. How bundling proteins interfere with MT sliding is unclear. In bipolar MT bundles in fission yeast, we found that the bundler ase1p localized all along the length of antiparallel MTs, whereas the motor klp2p (kinesin-14) accumulated only at MT plus ends. Consequently, sliding forces could only overcome resistant bundling forces for short, newly nucleated MTs, which were transported to their correct position within bundles. Ase1p thus regulated sliding forces based on polarity and overlap length, and computer simulations showed these mechanisms to be sufficient to generate stable bipolar bundles. By combining motor and bundling proteins, cells can thus dynamically organize stable regions of overlap between cytoskeletal filaments.  相似文献   

5.
The mechanism of formation of the negatively stained image in electron microscopy was infestigated with native collagen fibrils as a model. The negatively stained image was simulated from the primary structure by using the values of volume or bulkiness of each amino acid residue as a parameter for stain-excluding capacity. The pattern simulated from the bulkiness values gave an excellent fit with the negatively stained image. Since some contribution of positive staining components to negative staining has been suggested, positive staining with uranyl acetate was tested with various washing solutions of different pH. While acidic conditions did not produce any stained image, a positively stained image was easily obtained at alkaline pH. On the other hand, negatively stained images with stains of different charge character remained essentially the same as those obtained with acidic uranyl stains. It was concluded that the contribution of positive components to the negatively stained image is negligible under the conventional conditions for negative staining with uranyl acetate. In order to demonstrate the utility of the analytical method employing the values of "bulkiness," we studied the unknown molecular packing in the polar lead paracrystal of rabbit skeletal tropomyosin. Utilizing the primary sequence data for alpha-tropomyosin we successfully showed the polar paracrystal to be an array of molecules which are parallel and in register. Further, our analysis made it possible to deduce the position of a given residue in the negatively stained pattern of the polar paracrystal.  相似文献   

6.
Aggregates of paramyosin precipitated with divalent cations show a variety of forms with simple staining patterns and a period of 725 Å in the electron microscope. All the forms may be generated from a basic polar array where the molecules do not bond end-to-end: dark staining regions represent “gap” areas where stain can penetrate the paracrystal; light regions are “overlap” areas where stain is largely excluded. The various polar and dihedral arrays can be accounted for by a superposition of two of the basic polar arrays. This interpretation is supported by the appearance of “corrugation” at the lateral edge of the overlap regions of the fibers, by positive staining experiments, and by the “fringe” length observed at the ends of tactoids.  相似文献   

7.
Angle-layered aggregates of F-actin are net-like structures induced by Mg2+ concentrations below that used to form paracrystals. These aggregates incorporate the angular disorder of subunits, which has been described elsewhere for isolated actin filaments. Because this disorder is incorporated into the aggregates in solution at the time they are formed, the possibility of negative stain preparation being responsible for the disorder is excluded. The simple two-layered geometry of the angle-layered aggregate provides information about the shape of the component actin filaments free from the superposition of large numbers of layers. A model for the filament shape, derived from single filaments and confirmed by the angle-layered aggregate, is different from those that have previously emerged from paracrystal studies. An understanding of the interfilament bond in both the angle-layered aggregate and the paracrystal allows one to reconcile these different models. We have found a bipolar bonding rule, with staggered crossover points in the angle-layered aggregate, which we suggest is also responsible for Mg2+ paracrystals. This bonding rule can explain the apparent alignment of crossover points in adjacent filaments in paracrystals as a consequence of the superposition of staggered filaments.  相似文献   

8.
We investigated the mechanism by which meiotic spindles become bipolar and the correlation between bipolarity and poleward flux, using Xenopus egg extracts. By speckle microscopy and computational alignment, we find that monopolar sperm asters do not show evidence for flux, partially contradicting previous work. We account for the discrepancy by describing spontaneous bipolarization of sperm asters that was missed previously. During spontaneous bipolarization, onset of flux correlated with onset of bipolarity, implying that antiparallel microtubule organization may be required for flux. Using a probe for TPX2 in addition to tubulin, we describe two pathways that lead to spontaneous bipolarization, new pole assembly near chromatin, and pole splitting. By inhibiting the Ran pathway with excess importin-alpha, we establish a role for chromatin-derived, antiparallel overlap bundles in generating the sliding force for flux, and we examine these bundles by electron microscopy. Our results highlight the importance of two processes, chromatin-initiated microtubule nucleation, and sliding forces generated between antiparallel microtubules, in self-organization of spindle bipolarity and poleward flux.  相似文献   

9.
1. The TN-T and TN-I components of troponin both interact with tropomyosin and cause its precipitation in 0.1 M KC1 at neutral pH. The precipitate contains both end-to-end and side-by-side aggregates of tropomyosin molecules. 2. The TN-T and TN-I components change the band pattern of tropomyosin paracrystals formed in MgC1(2) solutions, although in different ways. TN-T causes the formation of hexagonal net structures, double-stranded net or paracrystals which result from the collapse of the double-stranded net. TN-I at pH 7.9 causes the formation of paracrystals with a 400 A periodic band pattern and a 200 A repeat. The same band pattern can also be seen in tropomyosin paracrystals formed at pH values below 6.0. 3. The TN-C component does not precipitate tropomyosin in 0.1 M KC1. The aggregates of tropomyosin obtained with either TN-T or TN-I can be solubilized by the addition of TN-C. No interaction of TN-C was observed with tropomyosin paracrystals formed in the presence of MgC12.  相似文献   

10.
We have used three different monoclonal antibodies (LCK16, JLH2 and JLF15) to tropomyosin for the localization of tropomyosin molecules within smooth muscle thin filaments. Thin filaments were incubated with monoclonal antibodies and visualized by negative staining electron microscopy. All three monoclonal antibodies caused the aggregation of thin filaments into ordered bundles, which displayed cross-striations with a periodicity of 37 ± 1 nm. In contrast, conventional rabbit antiserum to tropomyosin distorted and aggregated the thin filaments without generating cross-striations. Therefore, monoclonal antibodies to tropomyosin allow us, for the first time, to observe directly the distribution of tropomyosin molecules along the thin filaments of smooth muscle cells. The binding sites of the antibodies to skeletal muscle tropomyosin were examined by decorating tropomyosin paracrystals with monoclonal antibodies. The LCK16 monoclonal antibody binds the narrow band of tropomyosin paracrystals, whereas the JLF15 antibody binds the wide band of tropomyosin paracrystals.  相似文献   

11.
We examined the axial repeats in electron micrographs of three types of negatively stained paracrystals (two tactoid- and one sheet-like type) of rabbit light meromyosin (LMM) and its complex with C-protein characterized previously by similar axial period of about 43.0 nm. Assuming for the axial repeat in type II tactoids the value of 42.93 +/- 0.05 nm as it was determined by X-ray diffraction technique (Yagi and Offer 1981), we found average axial repeats in type I tactoid and in sheet-like paracrystal of 42.93 +/- 0.75 nm and 43.50 +/- 0.62 nm respectively. Analyzing the micrographs where the two types paracrystals are located side-by-side we determined rather accurately the average ratio of axial repeat in sheet-like paracrystal to that in type I tactoid (1.013 +/- 0.002). Taking 42.93 nm as the axial repeat in type I tactoid, the axial repeat in sheet-like paracrystal was found to be 43.50 +/- 0.08 nm. C-protein binds to LMM with the period of the underlying LMM paracrystals and independently of the value of their axial repeats. Two different axial repeats (42.9 nm and 43.5 nm) revealed for LMM paracrystals in this study precisely coincide with the average repeat periods of myosin crossbridges along the thick filaments found for different physiological states of skeletal muscles (Lednev and Kornev 1987). Molecular basis for the appearance of two structural states in LMM paracrystals and in the shafts of thick filaments are discussed.  相似文献   

12.
We have used internal deletions of multiples of seven residues to change the phase of the 28-residue charge repeat in a light meromyosin cDNA construct expressed in Escherichia coli. The solubility behaviour of these mutants was similar to that of the wild-type material, but the molecular packing in the aggregates formed at low ionic strength was different. Whereas wild-type material formed paracrystals in which molecules were in close contact over most of their length, molecules in the paracrystals formed by the mutants were in close contact for only a short distance, which was just short enough to exclude the deletion from the overlap. These data indicate that, although the 28-residue charge periodicity is important in myosin molecular interactions, it is probably not the major driving force for myosin assembly and instead influences the detailed axial stagger of the interacting molecules.  相似文献   

13.
Porcine platelet tropomyosin has been isolated by hydroxyapatite chromatography following isoelectric precipitation and ethanol fractionation. A single component (Mr = 30,000 on polyacrylamide gel electrophoresis in sodium dodecyl sulphate) was obtained in a variety of gel electrophoretic systems, including urea/sodium dodecyl sulphate and isoelectric focussing, suggesting the presence of a single polypeptide species. This contrasts with the observations by others using horse platelet tropomyosin or tropomyosins isolated from brain, where two polypeptides were found. The amino acid composition of porcine platelet tropomyosin was virtually identical to that of the horse platelet protein except for the presence of a single cysteine residue in the pig protein, whereas horse tropomyosin contains two. Oxidation of this sulphydryl group produced a molecule of over 60,000 Mr on polyacrylamide gels in sodium dodecyl sulphate, suggesting by analogy with skeletal muscle tropomyosin that the two chains had been linked and therefore existed in register in the coiled-coil structure. Cleavage at the cysteine produced a fragment of Mr = 19,000, indicating that this residue was located about one-third of the distance from a molecular end.Magnesium paracrystals of platelet tropomyosin were examined by electron microscopy following negative staining and found to have a repeat of 345 Å, close to that expected for an extended alpha-helical coiled-coil with an apparent Mr of 30,000. The repeating unit was centrosymmetric and the appearance of broken paracrystals suggested that the molecular ends lie on a dyad axis. Location of the sulphydryl residues in the paracrystals by labelling with N-pyrrolo-isomaleimide showed two bands separated by approximately 80 Å, which was consistent with the location of the molecular ends on a dyad.The amount of tropomyosin present was estimated as 2·2% of the total platelet protein. This implied a molar ratio of G-actin to tropomyosin of about 14:1, based on previous estimates of the actin content in porcine platelets. Assuming that one molecule of tropomyosin will bind six molecules of actin (based on the reduced molecular length of the platelet protein), there was not sufficient tropomyosin to bind to more than half the total actin in the cell.  相似文献   

14.
H-protein is a component of the thick filaments of skeletal myofibrils. Its effects on the assembly of myosin into filaments and on the formation of light meromyosin (LMM) paracrystals at low ionic strength have been investigated. H-protein reduced the turbidities of myosin filament and LMM paracrystal suspensions. Electron microscopic observation showed that the appearances of the filaments prepared in the presence and absence of H-protein were different. The filament length was not substantially changed by H-protein, but the diameter of the myosin filament was markedly reduced. H-protein bound to LMM and co-sedimented with it at low ionic strength upon centrifugation. Two types of paracrystals, spindle-shaped and sheet-like, were observed in LMM suspensions. H-protein altered the structure of the LMM paracrystals, especially the spindle-shaped ones. The thickness of the spindle-shaped paracrystals was reduced when H-protein was present during LMM paracrystal formation. On the other hand, periodic features along the long axis of the sheet-like paracrystals were retained even at high ratios of H-protein to LMM. However, there were fewer sheet-like paracrystals in the LMM suspensions containing H-protein than in the control. These results suggest that H-protein interferes with self-association of myosin molecule into filaments due to its binding to the tail portion of the myosin. However, H-protein does not have a length-determining effect on the formation of myosin filaments.  相似文献   

15.
Short regions of overlap between ends of antiparallel microtubules are central elements within bipolar microtubule arrays. Although their formation requires motors, recent in vitro studies demonstrated that stable overlaps cannot be generated by molecular motors alone. Motors either slide microtubules along each other until complete separation or, in the presence of opposing motors, generate oscillatory movements. Here, we show that Ase1, a member of the conserved MAP65/PRC1 family of microtubule-bundling proteins, enables the formation of stable antiparallel overlaps through adaptive braking of Kinesin-14-driven microtubule-microtubule sliding. As overlapping microtubules start to slide apart, Ase1 molecules become compacted in the shrinking overlap and the sliding velocity gradually decreases in a dose-dependent manner. Compaction is driven by moving microtubule ends that act as barriers to Ase1 diffusion. Quantitative modelling showed that the molecular off-rate of Ase1 is sufficiently low to enable persistent overlap stabilization over tens of minutes. The finding of adaptive braking demonstrates that sliding can be slowed down locally to stabilize overlaps at the centre of bipolar arrays, whereas sliding proceeds elsewhere to enable network self-organization.  相似文献   

16.
Correlation of tropomyosin amino acid sequence with uranyl acetate stained magnesium salt tactoids has been carried out by a computer graphic technique. Contrary to some previous suggestions, the results seem to indicate that there is no chain stagger within the molecule and the molecules are aligned with 178 ± 2 residue overlap in antiparallel array. In addition, the molecules for one particular tactoid appear to have an end overlap of approximately 21 residues.  相似文献   

17.
We have expressed in Escherichia coli a fragment of c-DNA that broadly corresponds to the alpha-helical coiled-coil rod section of glial fibrillary acidic protein (GFAP) and have used the resultant protein to prepare paracrystals in which molecular interactions can be investigated. An engineered fragment of mouse GFAP c-DNA was inserted into a modified version of the E. coli expression vector pLcII, from which large quantities of a lambda cII-GFAP rod fusion protein were prepared. A protein fragment corresponding to the GFAP rod was then obtained by proteolysis with thrombin. Paracrystals of this material were produced using divalent cations (Mg, Ca, Ba) in the presence of a chaotrophic agent such as thiocyanate. These paracrystals showed a number of polymorphic patterns that were based on a fundamental pattern that had dyad symmetry and an axial repeat of 57 nm. Analysis of both positive and negative staining patterns showed that this fundamental pattern was consistent with a unit cell containing two 48-nm-long molecules in an antiparallel arrangement with their NH2 termini overlapping by approximately 34 nm. More complicated patterns were produced by stacking the fundamental pattern with staggers of approximately 1/5, 2/5, and 1/2 the axial repeat. The molecular packing the unit cell was consistent with a range of solution studies on intermediate filaments that have indicated that a molecular dimer (i.e., a tetramer containing four chains or two coiled-coil molecules) is an intermediate in filament assembly. Moreover, these paracrystals allow the molecular interactions involved in the tetramer to be investigated in some detail.  相似文献   

18.
Crystallization of sickle cell hemoglobin proceeds by distinctive pathways which depend upon the pH and the ionic composition of the crystallizing milieu. The pathways differ in that after fibers form they associate into different intermediates which then crystallize. We term the pathways “high pH” and “low pH”. The value of the transition pH between the high pH and low pH pathways depends upon the specific ionic species present in the hemoglobin solution. Over the pH range studied the mechanism of crystallization is pH-dependent but the structure of the crystals ultimately formed is not.In this paper we describe two newly discovered intermediates involved in the crystallization of deoxyhemoglobin S via the low pH pathway. The first of these consists of a class of particles we call macrofibers. Optical diffraction patterns of fibers and macrofibers have similar intensity distributions and layer-line spacings suggesting that macrofibers and fibers are assembled from a common structural unit which we take to be the Wishner-Love double strand.The second new structure is a paracrystalline form of deoxyhemoglobin S. The paracrystal is built from layers of double strands of molecules in an arrangement similar to that within the crystals. Optical diffraction of electron micrographs of paracrystals reveals that longitudinal disorder is present between double strands. Projections of the electron density down the c axis of the crystal provide images very similar to those in electron micrographs of negatively stained paracrystals. The patterns appearing in the paracrystal due to the disorder can be fully simulated by shifts between the layers of double strands.  相似文献   

19.
Polar zippers     
BACKGROUND: Certain proteins are known to form leucine zippers - alpha-helical coiled-coils in which the non-polar side chains of two leucine-rich helices intermesh. We recently presented the first evidence for a polar zipper, formed by the carboxy-terminal peptides of the eight subunits of Ascaris haemoglobin. The evidence was based on the presence of pairs of acidic residues alternating with pairs of basic residues ( + + - - ) in an amino-acid sequence that has since been shown to be incomplete. The complete sequence, derived from the haemoglobin's cDNA, now shows a self-complementary polar sequence extending along the entire length of its 24-residue carboxy-terminal peptide. RESULTS: From the complete sequence, it is clear that the eight identical subunits of the haemoglobin could be held together by an eight-stranded antiparallel beta barrel made up of the carboxy-terminal 24 residues of each of the subunits, such that each strand forms 10 salt bridges with each of its neighbours. A computer search of the protein database revealed similar, but shorter, + + - - repeats in several other proteins. It also revealed long repeats of alternating arginine and aspartate residues, and long stretches of only glutamines, or only serines, suggestive of several other kinds of polar zippers. CONCLUSION: Several proteins have amino-acid sequences that suggest the formation of polar zippers made of beta strands. These could form antiparallel pleated sheets linked together by hydrogen bonds between polar side chains both above and below the plane of the sheets. Polar zippers may be important in welding together oligomeric proteins which have subunits lacking the extensive complementary surfaces necessary for stability, or in promoting the association of functionally complementary proteins.  相似文献   

20.
A refined computer graphics approach to correlation of molecular sequence with electron micrographs of tropomyosin tactoids is presented. It is shown that antiparallel structures with molecular chains in phase, 21 or 14 residue overlap and C or N terminal overlap are consistent with the morphology. The C terminal overlap structure previously postulated gives the best direct correlation but chemical evidence appears to support the N terminal overlap structure. The parallel tactoid form appears more complicated and no adequate structure has yet been elucidated.  相似文献   

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