Structural intermediates in the assembly of taxoid-induced microtubules and GDP-tubulin double rings: time-resolved X-ray scattering

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

Conformational changes in bacteriophage Ø 29 connector prevents DNA-binding activity*

In vitro DNA packaging activity in a defined system derived from bacteriophage Ø29 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

X-Ray Diffraction Studies of Whole Rat Heart During Anoxic Perfusion

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 (I1,0 /I1,1) 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

The Effect of Temperature on the Structure of Vinblastine-induced Polymers of Purified Tubulin: Detection of a Reversible Conformational Change

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 microtubulesin vitrounder normal conditions of assembly

Crystallization of nanoscale-confined diblock copolymer chains

Crystallization of polymer chains between hard glassy walls or between amorphous domains in a nanoscale lamellar structure has been observed using simultaneous small-angle and wide-angle X-ray scattering (SAXS/WAXS). Semicrystalline symmetric diblock copolymers containing poly(ethylene) (PE) and a room-temperature glassy or amorphous component were shear oriented in the high temperature lamellar melt, then quenched below the PE melt temperature. For the glassy sample, the orientation of chain-folded PE stems was deduced from SAXS/WAXS peak positions to be parallel to the lamellar interface. Diffuse scattering bars consistent with lateral positional correlations of the PE crystallites were observed only in the SAXS patterns for the glassy sample with the X-rays incident parallel to the lamellae. In contrast, in a sample containing amorphous lamellae, PE crystallization occurred with weak crystallite orientation and no lateral positional correlations of crystallites

W16.1: A new fixed wavelength diffraction station at the SRS Daresbury

Station W16.1 is a fixed wavelength (1.4 Å) x‐ray diffraction station recently constructed and commissioned at the SRS. It has been designed specifically for time‐resolved studies of noncrystalline and fibrous materials and optimized for low angle measurements. Wide angle diffraction will also be available with simultaneous small and wide angle scattering/diffraction a future facility. In order to perform dynamic (∼1 ms) low angle measurements on weakly scattering systems, the station design has had to incorporate several novel features so as to achieve the predicted 1×1013 photon/s at the specimen