ABSTRACT
Semisolid liquid paraffin-in-water emulsions (aqueous creams) prepared from cetrimide/fatty alcohol mixed emulsifiers, and ternary systems formed by dispersing the mixed emulsifier in controlled percentages of water were examined as they aged using a combination of low and high angle X-ray diffraction measurements (Daresbury Laboratory Synchrotron Radiation Source). The results were correlated with the rheological properties measured in earlier studies. The cationic emulsifying wax showed phenomenal swelling in water. The reflection that incorporates interlamellar water increased continuously from 74 A at 28% water to over 500 A at 93% water. The trend was not influenced by the method of incorporation of the components and swollen lamellar phase was also identified in the corresponding emulsion. The swelling, which was due to electrostatic repulsion, was suppressed by salt and was reduced when the surfactant counterion was changed from Br(-) to Cl(-). Changes in rheological properties on storage and in the presence of salt were correlated with changes in water layer thickness. High angle diffraction confirmed that the hydrocarbon bilayers were in the hexagonal alpha-crystalline mode of packing. Ternary systems and creams prepared from pure alcohols, although initially semisolid, were rheologically unstable and broke down. Low angle X-ray study into the kinetics of structure breakdown showed that the swollen lamellar gel phase formed initially swells even further on storage before separating.
Subject(s)
Cetrimonium Compounds/administration & dosage , Fatty Alcohols/administration & dosage , Ointments , Emulsions , X-Ray DiffractionABSTRACT
Small-angle X-ray diffraction data has been collected from 99 ;core-cut' breast tissue specimens representing a number of different pathologies. Data in the range 75-1390 A have been compared with controls from patients with no breast disease. Bessel functions and Bragg maxima resulting from the fibrillar structure of collagen have been identified. The Bragg maxima indexed onto a 649 A lattice. Systematic differences in the intensities and D-spacings between the collagen of malignant, benign and normal tissue groups have been clearly demonstrated and quantified. These differences appear to be due to a significantly lower structural order within the malignant tissues. Possible explanations for this are discussed and the potential for utilizing this observation in cancer diagnosis is considered.
ABSTRACT
We report the first time-resolved study of the two-dimensional x-ray diffraction pattern during active contraction in insect flight muscle (IFM). Activation of demembranated Lethocerus IFM was triggered by 1.5-2.5% step stretches (risetime 10 ms; held for 1.5 s) giving delayed active tension that peaked at 100-200 ms. Bundles of 8-12 fibers were stretch-activated on SRS synchrotron x-ray beamline 16.1, and time-resolved changes in diffraction were monitored with a SRS 2-D multiwire detector. As active tension rose, the 14.5- and 7.2-nm meridionals fell, the first row line dropped at the 38.7 nm layer line while gaining a new peak at 19.3 nm, and three outer peaks on the 38.7-nm layer line rose. The first row line changes suggest restricted binding of active myosin heads to the helically preferred region in each actin target zone, where, in rigor, two-headed lead bridges bind, midway between troponin bulges that repeat every 38.7 nm. Halving this troponin repeat by binding of single active heads explains the intensity rise at 19.3 nm being coupled to a loss at 38.7 nm. The meridional changes signal movement of at least 30% of all myosin heads away from their axially ordered positions on the myosin helix. The 38.7- and 19.3-nm layer line changes signal stereoselective attachment of 7-23% of the myosin heads to the actin helix, although with too little ordering at 6-nm resolution to affect the 5.9-nm actin layer line. We conclude that stretch-activated tension of IFM is produced by cross-bridges that bind to rigor's lead-bridge target zones, comprising < or = 1/3 of the 75-80% that attach in rigor.
Subject(s)
Actins/metabolism , Actins/ultrastructure , Muscle Contraction/physiology , Muscle Fibers, Skeletal/ultrastructure , Myosins/metabolism , Myosins/ultrastructure , Animals , Flight, Animal , Hemiptera , In Vitro Techniques , Models, Biological , Models, Chemical , Muscle, Skeletal/physiology , Time Factors , X-Ray DiffractionABSTRACT
We have studied the self-association reactions of purified GDP-liganded tubulin into double rings and taxoid-induced microtubules, employing synchrotron time-resolved x-ray solution scattering. The experimental scattering profiles have been interpreted by reference to the known scattering profiles to 3 nm resolution and to the low-resolution structures of the tubulin dimer, tubulin double rings, and microtubules, and by comparison with oligomer models and model mixtures. The time courses of the scattering bands corresponding to the different structural features were monitored during the assembly reactions under varying biochemical conditions. GDP-tubulin essentially stays as a dimer at low Mg(2+) ion activity, in either the absence or presence of taxoid. Upon addition of the divalent cations, it associates into either double-ring aggregates or taxoid-induced microtubules by different pathways. Both processes have the formation of small linear (short protofilament-like) tubulin oligomers in common. Tubulin double-ring aggregate formation, which is shown by x-ray scattering to be favored in the GDP- versus the GTP-liganded protein, can actually block microtubule assembly. The tubulin self-association leading to double rings, as determined by sedimentation velocity, is endothermic. The formation of the double-ring aggregates from oligomers, which involves additional intermolecular contacts, is exothermic, as shown by x-ray and light scattering. Microtubule assembly can be initiated from GDP-tubulin dimers or oligomers. Under fast polymerization conditions, after a short lag time, open taxoid-induced microtubular sheets have been clearly detected (monitored by the central scattering and the maximum corresponding to the J(n) Bessel function), which slowly close into microtubules (monitored by the appearance of their characteristic J(0), J(3), and J (n) - (3) Bessel function maxima). This provides direct evidence for the bidimensional assembly of taxoid-induced microtubule polymers in solution and argues against helical growth. The rate of microtubule formation was increased by the same factors known to enhance taxoid-induced microtubule stability. The results suggest that taxoids induce the accretion of the existing Mg(2+)-induced GDP-tubulin oligomers, thus forming small bidimensional polymers that are necessary to nucleate the microtubular sheets, possibly by binding to or modifying the lateral interaction sites between tubulin dimers.
Subject(s)
Guanosine Diphosphate/metabolism , Microtubules/physiology , Paclitaxel/analogs & derivatives , Paclitaxel/pharmacology , Taxoids , Tubulin/metabolism , Animals , Antineoplastic Agents, Phytogenic/pharmacology , Brain/metabolism , Cattle , Docetaxel , Guanosine Triphosphate/metabolism , Kinetics , Microtubules/drug effects , Microtubules/ultrastructure , Models, Structural , Time Factors , Tubulin/drug effects , Tubulin/isolation & purification , X-Ray DiffractionABSTRACT
A system has been developed which represents a significant advance in the quality and extent of small- and wide-angle X-ray scattering data (SAXS and WAXS) that can be recorded simultaneously with strain data during the drawing and annealing of polymer materials. WAXS data are recorded using a Photonic Science charge-coupled-device area detector and SAXS data using a gas-filled multiwire area detector. Strain data, for the region of the specimen from which the SAXS/WAXS data are collected, are calculated from an accurately synchronized continuously recorded video image of the specimen. The system allows X-ray and video image data to be collected as a series of frames with essentially no ;dead-time' between frames. The data are fully two-dimensional and can be collected for a wide range of d spacings. The use of this system to investigate the stress-induced orientation and phase changes during the drawing of a range of grades of commercially available polyethylene is described.
ABSTRACT
Addition of the antimitotic drug vinblastine to solutions of purified tubulin induces the formation of helical polymers whose structure and type of aggregation is determined by the concentration of magnesium. While paracrystalline arrangements of single coils are observed at low concentrations of the ion, for concentrations higher than 6 mM free double-coiled spirals are obtained, which are indistinguishable from those obtained in the presence of microtubule-associated proteins (MAPs). This result is consistent with a similar effect of magnesium and MAPs in neutralizing negative charges on the tubulin molecule and so allowing for lateral contacts between protofilaments. The effects that temperature has on the structure of both types of polymers, free spirals or paracrystals, have been monitored using time-resolved X-ray solution scattering. This study shows that a temperature increase: (1) affects the length and lateral aggregation of the spirals in the paracrystalline sample; (2) induces a reversible increase of the helical pitch in both types of polymers that closely follows the temperature change; (3) produces an irreversible aggregation of some of the protein in both types of polymers; and (4) can induce a reversible transformation from one type of structure to the other when the concentration of Mg2+ is in the boundary between the two ranges. We suggest that the changes in pitch are due to a temperature-induced conformational change of the tubulin molecule. This effect may be related to the structural modifications that result in the temperature-induced assembly of microtubules in vitro under normal conditions of assembly.
Subject(s)
Tubulin/chemistry , Vinblastine/pharmacology , Animals , Brain Chemistry , Magnesium/pharmacology , Models, Molecular , Polymers , Protein Conformation , Scattering, Radiation , Solutions , Swine , Temperature , Tubulin/drug effects , Tubulin/isolation & purification , Tubulin/ultrastructure , X-Ray Diffraction , X-RaysABSTRACT
Using synchrotron radiation and whole muscles, 2 ms time-resolved x-ray diffraction patterns were recorded at 8 degrees C. The results show that in both isotonic and isometric contractions, as well as in length changes imposed at maximum tension [Po], the meridional third myosin layer line consists of two distinct reflections with different intensities and spacings that measure approximately 14,623 and 14,412 nm at Po. Although the intensity behavior of the two reflections is strikingly different during quick releases, it is very similar during stretches. Study of the time courses indicates that myosin heads diffracting at Po with the approximately 14.623 nm periodicity are actively involved in tension production. Those diffracting at Po with the periodicity of approximately 14.412 nm appear not be associated with tension production during isometric contraction and releases, but the results suggest that they are recruited during stretches and here contribute to tension production. Our most important conclusion is that under all conditions of contraction we have investigated there exist two populations of myosin heads, each with a well defined axial disposition and configuration.
Subject(s)
Muscle Contraction/physiology , Muscles/chemistry , Myosins/chemistry , Animals , Biophysical Phenomena , Biophysics , Elasticity , Electric Stimulation , In Vitro Techniques , Isometric Contraction/physiology , Muscles/physiology , Myosins/physiology , Rana catesbeiana , X-Ray DiffractionABSTRACT
The synchrotron x-ray solution scattering profiles of microtubules assembled from purified GDP- or GTP-tubulin with the antitumor drug docetaxel (Taxotere) are consistent with identical non-globular alpha and beta-tubulin monomers ordered within the known surface lattice of microtubules, with a center to center lateral spacing of 5.7 +/- 0.1 nm. The higher angle part of the scattering profile, and therefore the substructure of the microtubule wall is identical in Taxotere- and Taxol-induced microtubules, to the resolution of the measurements. However, Taxotere-induced microtubules have a mean diameter of 24.2 +/- 0.4 nm, which is 1.12 +/- 0.01 times larger than that of paclitaxel (Taxol) induced microtubules. The population of Taxotere microtubules has on average 13.4 protofilaments, which is similar to control microtubules assembled with glycerol but is in marked contrast with Taxol-induced microtubules, which have on average 12 protofilaments under identical solution conditions. Model populations of Taxotere and Taxol microtubules with the distributions of protofilament numbers determined by electron microscopy reproduce the positions and approximate intensities of the experimental x-ray scattering data. Comparison of the structures and activities of both taxoids strongly suggests that the change of the more frequent lateral bond angle between tubulin molecules from 152.3 degrees (13-protofilament microtubules) to 150 degrees (12-protofilament microtubules) is linked to the binding of the side chain of Taxol. Optimal microtubule formation is obtained with unitary Taxotere to tubulin heterodimer ratio; however, ligand molecules in excess over tubulin dimers cause a loss of cylindrical scattering features, consistent with microtubule opening. The results are compatible with the observed biochemical and thermodynamic properties of this ligand-induced microtubule assembly system and also with the simple working hypothesis that taxoids would bind between adjacent microtubule protofilaments.
Subject(s)
Microtubules/ultrastructure , Paclitaxel/analogs & derivatives , Taxoids , Docetaxel , Guanosine Triphosphate/pharmacology , In Vitro Techniques , Microscopy, Electron , Microtubules/drug effects , Models, Molecular , Paclitaxel/chemistry , Paclitaxel/pharmacology , Protein Binding , Scattering, Radiation , Solutions , Synchrotrons , Tubulin/metabolism , X-RaysABSTRACT
Equatorial x-ray reflections were recorded from the ventricular region of whole rat heart, during the transition from normoxia to anoxia. The intensity ratio of the 1.0 and 1.1 equatorial reflections (I 1.0/I 1.1) was 2.96 during normoxic perfusion, decreasing to 0.37 after 115 minutes of anoxic perfusion and closely parallel those reported between partially dissected relaxed and rigor vertebrate heart muscle (1.2). The peak positions of these reflections both increased by ca. 5% during anoxic perfusion indicative of a lateral expansion in the sarcomere filament lattice. These results indicate that the process of anoxia leads to the condition of rigor in which the majority of the myosin cross-bridges bind to the thin filament. Using this technique global changes in whole heart structure can be studied, and due to the ease of perfusion of the heart, biochemical and physiological problems may be investigated in relation to the structure of the heart as a whole. This may be of clinical interest, particularly in terms of investigations into organ preservation and transplantation. This is believed to be the first occasion where intact muscular organs have been studied in this way.
Subject(s)
Hypoxia/metabolism , Myocardium/chemistry , Animals , Hypoxia/pathology , In Vitro Techniques , Male , Myocardium/pathology , Perfusion , Rats , Rats, Wistar , X-Ray DiffractionABSTRACT
Using the facilities at the Daresbury Synchrotron Radiation Source, meridional diffraction patterns of muscles at ca 8 degrees C were recorded with a time resolution of 2 or 4 ms. In isometric contractions tetanic peak tension (P0) is reached in ca 400 ms. Under such conditions, following stimulation from rest, the timing of changes in the major reflections (the 38.2 nm troponin reflection, and the 21.5 and 14.34/14.58 nm myosin reflections) can be explained in terms of four types of time courses: K1, K2, K3 and K4. The onset of K1 occurs immediately after stimulation, but that of K2, K3 and K4 is delayed by a latent period of ca 16 ms. Relative to the end of their own latent periods the half-times for K1, K2, K3 and K4 are 14-16, 16, 32 and 52 ms, respectively. In half-times, K1, K2, K3 lead tension rise by 52, 36 and 20 ms, respectively. K4 parallels the time course of tension rise. From an analysis of the data we conclude that K1 reflects thin filament activation which involves the troponin system; K2 arises from an order-disorder transition during which the register between the filaments is lost; K3 is due to the formation of an acto-myosin complex which (at P0) causes 70% or more of the heads to diffract with actin-based periodicities; and K4 is caused by a change in the axial orientation of the myosin heads (relative to thin filament axis) which is estimated to be from 65-70 degrees at rest to ca 90 degrees at P0. Isotonic contraction experiments showed that during shortening under a load of ca 0.27 P0, at least 85% of the heads (relative to those forming an acto-myosin complex at P0) diffract with actin-based periodicities, whilst their axial orientation does not change from that at rest. During shortening under a negligible load, at most 5-10% of the heads (relative to those forming an acto-myosin complex at P0) diffract with actin-based periodicities, and their axial orientation also remains the same as that at rest. This suggests that in isometric contractions the change in axial orientation is not the cause of active tension production, but rather the result of it. Analysis of the data reveals that independent of load, the extent of asynchronous axial motions executed by most of the cycling heads is no more than 0.5-0.65 nm greater than at rest.(ABSTRACT TRUNCATED AT 400 WORDS)
Subject(s)
Isometric Contraction/physiology , Isotonic Contraction/physiology , Muscle, Skeletal/chemistry , Muscle, Skeletal/physiology , Myosins/analysis , Myosins/physiology , Rana catesbeiana/physiology , Animals , Mathematics , Myosins/chemistry , Time Factors , X-Ray DiffractionABSTRACT
Results were obtained from contracting frog muscles by collecting high quality time-resolved, two-dimensional, X-ray diffraction patterns at the British Synchrotron Radiation Source (SERC, Daresbury, Laboratory). The structural transitions associated with isometric tension generation were recorded under conditions in which the three-dimensional order characteristic of the rest state is either present or absent. In both cases, new layer lines appear during tension generation, subsequent to changes from activation events in the thin filaments. Compared with the 'decorated' actin layer lines of the rigor state, the spacings of the new layer lines are similar whereas their intensities differ substantially. We conclude that in contracting muscle an actomyosin complex is formed whose structure is not like that in rigor, although it is possible that the interacting sites are the same. Transition from rest to plateau of tension is accompanied by approximately 1.6% increase in the axial spacing of the myosin layer lines. This is explained as arising from axial disposition of the interacting myosin heads in the actomyosin complex. Model calculations are presented which support this view. We argue that in a situation where an actomyosin complex is formed during contraction, one cannot describe the diffraction features as being either thick or thin filament based. Accordingly, the layer lines seen during tension generation are referred to as actomyosin layer lines. It is shown that these layer lines can be indexed as submultiples of a minimum axial repeat of approximately 218.7 nm. After lattice disorder effects are taken into account, the intensity increases on the 15th and 21st AM layer lines at spacings of approximately 14.58 and 10.4 nm respectively, show the same time course as tension rise. However, the time course of the intensity increase of the other actomyosin layer lines and of the spacing change (which is the same for both phenomena) shows a substantial lead over tension rise. These findings suggest that the actomyosin complex formed prior to tension rise is a non-tension-generating state and that this is followed by a transition of the complex to a tension-generating state. The intensity increase in the 15th actomyosin layer line, which parallels tension rise, can be accounted for assuming that in the tension-generating state the attached heads adopt (axially) a more perpendicular orientation with respect to the muscle axis than is seen at rest or in the non-tension-generating state. This suggests the existence of at least two structurally distinct interacting myosin head conformations.(ABSTRACT TRUNCATED AT 400 WORDS)
Subject(s)
Actomyosin/ultrastructure , Isometric Contraction , X-Ray Diffraction/methods , Animals , Kinetics , Rana catesbeiana/physiology , Stress, MechanicalABSTRACT
The structure of microtubules has been characterized to 3 nm resolution employing time-resolved X-ray scattering. This has revealed detailed structural features of microtubules not observed before in solution. The polymerization of highly purified tubulin, induced by the antitumour drug taxol, has been employed as a microtubule model system. This assembly reaction requires Mg2+, is optimal at a 1:1 taxol to tubulin heterodimer molar ratio, proceeds with GTP or GDP and is intrinsically reversible. The X-ray scattering profiles are consistent with identical non-globular alpha and beta-tubulin monomers ordered within the known helical surface lattice of microtubules. Purified tubulin-taxol microtubules have a smaller mean diameter (approx. 22 nm) than those induced by microtubule associated proteins or glycerol (approx. 24 nm), but nearly identical wall substructure to the resolution of the measurements. This is because the majority of the former consist of only 12 protofilaments instead of the typical 13 protofilaments, as confirmed by electron microscopy of thin-sectioned, negatively stained and ice-embedded taxol microtubules. It may be concluded that taxol induces a slight reduction of the lateral contact curvature between tubulin monomers. The main fringe pattern observed in cryo-electron micrographs is consistent with a simple 12 protofilament 3-start skewed lattice model. Cylindrical closure of this lattice can be achieved by tilting the lattice 0.8 degrees with respect to the microtubule axis. The closure implies a discontinuity in the type of lateral contacts between the tubulin monomers (regardless of whether these are of the -alpha-beta- or the -alpha-alpha-/-beta-beta- type), which indicates that lateral contacts and the subunit specificity of taxol binding are, to a large degree, equivalent.
Subject(s)
Alkaloids/pharmacology , Microtubules/drug effects , Tubulin/metabolism , Glycerol , Guanine Nucleotides/metabolism , Magnesium/metabolism , Microscopy, Electron , Microtubule-Associated Proteins/metabolism , Microtubules/chemistry , Microtubules/ultrastructure , Paclitaxel , Scattering, Radiation , X-RaysABSTRACT
Small angle X-ray scattering (SAXS) with synchroton radiation was used to investigate interactions among lipid particles in lecithin-bile salt model systems and in native gallbladder biles. In model systems in the absence of cholesterol, isotropic, continuous spectra were found, indicating the absence of periodic structures. In the presence of excess cholesterol, interaction in the form of lamellar stacking was detected by the appearance of discrete diffraction peaks. In the supersaturated cholesterol region of the commonly accepted phase diagram [1], where cholesterol crystals were expected, we found lamellar stacking. The high proportion of cholesterol to bile salts seems to be the common denominator of these models. The lamellar stacking was also found in native unprocessed bile. This effect of cholesterol on lipid structure has not been previously described. Lamellar stacking may contribute to cholesterol solubilization. Its influence on the kinetics of cholesterol crystallization is presently unknown.
Subject(s)
Bile Acids and Salts , Bile/physiology , Cholesterol , Phosphatidylcholines , Humans , Particle Accelerators , X-Ray DiffractionABSTRACT
The acrosomal process of Limulus sperm contains a bundle of filaments composed of actin and a 102 kDa protein in a 1:1 molar ratio. The structure of the bundle in true discharge was investigated by electron cryomicroscopy, X-ray scattering and crystallographic image analysis. A bundle can be characterized as a quasi-crystal with continuously varying views along the bundle axis. Each segment of the bundle is found to obey the symmetry of space group P1, with a = b = 147 A, c = 762 A, alpha = 90 degrees, beta = 90.6 degrees, gamma = 120 degrees. A unit cell contains a helical repeat of the filament with a selection rule following that of an actin filament. A 24 A projection map based on the h0l view was reconstructed after averaging 5300 unit cells from six electron images. Filaments in this projection are well separated and clearly display a 21 screw symmetry. This screw symmetry results from the helical parameters of the bundle filament and is found to be a non-crystallographic symmetry element present in the unit cell. Our structural analysis has led to the proposal that the assembly of a stable bundle with a defined maximum diameter can be controlled by the crystallographic packing of the twisted filaments.
Subject(s)
Acrosome/chemistry , Actins/chemistry , Horseshoe Crabs/chemistry , X-Ray Diffraction , Acrosome/ultrastructure , Actins/isolation & purification , Actins/ultrastructure , Animals , Freeze Fracturing , Horseshoe Crabs/ultrastructure , Image Processing, Computer-Assisted , Male , Molecular Weight , Peptides/chemistry , Peptides/isolation & purificationABSTRACT
This report provides a preliminary sketch of the results obtained in a two-dimensional time resolved X-ray diffraction study of "live" frog sartorius muscles undergoing isometric tetani. These results demonstrate the recently developed capability to record time resolved (10 msec time resolution), two-dimensional X-ray diffraction diagrams throughout the cycle of contraction. The correlation between the time courses of the diffraction features in the whole of the diffraction diagram establishes a sequence of structural events, which suggest that during the transition from rest to the plateau of tension and the subsequent recovery, the following sequence of events takes place: a) Following the activation phase, which is best monitored by the increase of intensity on the second actin layer line at 18.0 nm spacing (5), there is the onset of three dimensional disorder due to the filaments losing their axial alignment and the myosin heads rotating azimuthally and moving radially outwards. A set of low-angle layer lines, following the actin based spacings seen in rigor (i.e., at spacings of ca. 36.5-37.5, 24.0 and 18.0 nm) become visible and those at ca. 24.0 and 18.0 nm appear to increase in intensity during this phase with a time course that cannot be determined accurately because of the proximity of the neighbouring first, second and third myosin layer lines and the weakness of these diffraction features. Whether the first of these layer lines increases or not is difficult to ascertain. Moreover, proper account of the loss in crystallinity during the development of tension must be made before the comparisons in intensity between the rest and peak of tension states have any significance. Nevertheless, these features together with the behaviour of the equatorial reflections and the meridional region of the third myosin layer line indicate that a sizeable fraction of the crossbridges may become axially disposed with an actin based periodicity. The formation of this complex does not immediately result in the generation of tension. The labelling of the thin filaments is also reflected in the main actin layer lines at 5.9 and 5.1 nm. b) The tension generating phase is monitored by the intensity changes in the meridional region of the third myosin layer line, which are best explained by a change in the orientation/conformation of the tension bearing crossbridges, (which probably adopt a more perpendicular orientation to the filament axis). c) At the end of stimulation, the crossbridges return to an axial spacing and axial orientation (although not yet azimuthal) similar to the one at rest.(ABSTRACT TRUNCATED AT 400 WORDS)
Subject(s)
Isometric Contraction , Muscles/physiology , Animals , In Vitro Techniques , Rana catesbeiana , X-Ray Diffraction/methodsABSTRACT
We have previously shown that time-resolved X-ray diffraction studies of the 2-D pattern from isometrically contracting flatfish (turbot) fin muscle have considerable advantages over similar studies of other vertebrate muscles due to the simple lattice and the better long-range order in these muscles (5, 24). Here we show not only that two structurally different myosin head to actin attached states must exist in the crossbridge cycle but that we are also able to define the likely crossbridge configurations in these states. A non-force producing "weak-binding" state is evident from the lead of the (11) equatorial reflections (and actin mass) time-course relative to that of tension (and the (10) equatorial reflection decrease) by about 30 msec. The first myosin layer line at 429 A has a weakened but altered intensity distribution, with no change in axial spacing, in patterns from active muscle. We show this to be consistent with myosin heads binding in the non-specific manner envisaged for the "weak-binding" state. Evidence for the second force-producing attached state, or series of states, comes from the observation of only a small increase in the intensity of the 360 A actin layer line between resting and active muscle patterns. It might be thought that a substantial increase in this layer line would be expected if myosin heads were even transiently attached to the thin filaments in a force-producing state. However, this is not so because internal changes in the structure of the thin filaments in active muscle have the opposite effect of causing this layer line to decrease in intensity. Observation of a small net intensity increase is therefore evidence for myosin head attachment with the symmetry of the actin helix. From the equatorial diffraction pattern, Fourier synthesis maps were computed at 10 msec intervals throughout the isometric tetanus, enabling changes in the mass distribution to be visualised between the force- and non-force producing populations of crossbridges. This difference map shows that in the force-producing state myosin heads have their centres of mass on average at a smaller radius from the thin filament axis compared to the case for non-force producing myosin heads. Since there is good evidence that there is no substantial change in myosin head shape during contraction (30) these observations are consistent with myosin heads swinging on actin as fairly rigid structures.
Subject(s)
Actins/physiology , Isometric Contraction , Muscles/physiology , Myosins/physiology , Actins/chemistry , Animals , Flatfishes , Flounder , In Vitro Techniques , Models, Structural , Myosins/chemistry , Particle Accelerators , X-Ray Diffraction/methodsABSTRACT
In vitro DNA packaging activity in a defined system derived from bacteriophage phi 29 depends upon the chemical integrity of the connector protein p10. Proteolytic cleavage of p10 rendered the proheads inactive for DNA packaging. A similar treatment on isolated connectors abolished the DNA-binding activity of the native p10, but the general shape and size of the connector was not changed as revealed by electron microscopy. Analytical ultracentrifugation showed that the proteolyzed connectors had a smaller sedimentation coefficient, while amino acid analysis after dialysis of the proteolyzed p10 confirmed the loss of 16 and 19 amino acids from the amino and carboxy termini, respectively. Low angle X-ray scattering revealed that proteolysis was followed by a small decrease in the radius of gyration and a reorganization of the distal domain of the cylindrical inner part of the connector. Characterization of the cleavage sites in the primary sequence allowed us to propose the location of the DNA-binding domain in the connector model.