The effect of thin filament activation on the attachment of weak binding cross-bridges: A two-dimensional x-ray diffraction study on single muscle fibers.
To study possible structural changes in weak cross-bridge attachment to actin upon activation of the thin filament, two-dimensional (2D) x-ray diffraction patterns of skinned fibers from rabbit psoas muscle were recorded at low and high calcium concentration in the presence of saturating concentrations of MgATPgammaS, a nucleotide analog for weak binding states. We also studied 2D x-ray diffraction patterns recorded under relaxing conditions at an ionic strength above and below 50 mM, because it had been proposed from solution studies that reducing ionic strength below 50 mM also induces activation of the thin filament. For this project a novel preparation had to be established that allows recording of 2D x-ray diffraction patterns from single muscle fibers instead of natural fiber bundles. This was required to minimize substrate depletion or product accumulation within the fibers. When the calcium concentration was raised, the diffraction patterns recorded with MgATPgammaS revealed small changes in meridional reflections and layer line intensities that could be attributed in part to the effects of calcium binding to the thin filament (increase in I380, decrease in first actin layer line intensity, increase in I59) and in part to small structural changes of weakly attached cross-bridges (e.g., increase in I143 and I72). Calcium-induced small-scale structural rearrangements of cross-bridges weakly attached to actin in the presence of MgATPgammaS are consistent with our previous observation of reduced rate constants for attachment and detachment of cross-bridges with MgATPgammaS at high calcium. Yet, no evidence was found that weakly attached cross-bridges change their mode of attachment toward a stereospecific conformation when the actin filament is activated by adding calcium. Similarly, reducing ionic strength to less than 50 mM does not induce a transition from nonstereospecific to stereospecific attachment. (+info)
Model studies of chromatin structure based on X-ray diffraction data.
Model calculations are presented in order to interpret the X-ray diffraction diagrams given by chromatin gels. It is shown that by taking into account the hydration of chromatin subunits, the problem of calculating the interference function in concentrated gels is greatly simplified. In this way it is spossible to fully interpret the influence of concentration on the position and intensity of the various rings present in the X-ray diffraction patterns. The possibilities and limitations of models based on spherical symmetry are also discussed. It is concluded that each chromatin subunit most likely contains three turns of DNA in each 200 base pairs segment surrounding a central protein core. With the method presented here it is possible to test if other models of chromatin based on different kinds of evidence are compatible with the X-ray diffraction data. (+info)
Chromatin nu bodies: isolation, subfractionation and physical characterization.
Monomer chromatin subunit particles (nu1) have been isolated in gram quantities by large-scale zonal centrifugation of micrococcal nuclease digests of chicken erythrocyte nuclei. nu1 can be stored, apparently indefinitely, frozen in 0.2 mM EDTA (pH 7.0) at less than or equal to 25 degrees C. Aliquots of the stored monomers have been subfractionated by dialysis against 0.1 M KCl buffers into a soluble fraction containing equimolar amounts of H4, H3, H2A, H2B associated with a DNA fragment of approximately 130-140 nucleotide pairs, and a precipitated fraction containing all of the histones including H5 and H1 associated with DNA fragments. The total nu1 and the KCl-soluble fraction of nu1 have been examined by sedimentation, diffusion, sedimentation equilibrium ultracentrifugation, low-angle X-ray diffraction, and electron microscopy. Physical parameters from all of these techniques are presented and correlated in this study. (+info)
Maximum solubility of cholesterol in phosphatidylcholine and phosphatidylethanolamine bilayers.
In any lipid bilayer membrane, there is an upper limit on the cholesterol concentration that can be accommodated within the bilayer structure; excess cholesterol will precipitate as crystals of pure cholesterol monohydrate. This cholesterol solubility limit is a well-defined quantity. It is a first-order phase boundary in the phospholipid/cholesterol phase diagram. There are many different solubility limits in the literature, but no clear picture has emerged that can unify the disparate results. We have studied the effects that different sample preparation methods can have on the apparent experimental solubility limit. We find that artifactual demixing of cholesterol can occur during conventional sample preparation and that this demixed cholesterol may produce artifactual cholesterol crystals. Therefore, phospholipid/cholesterol suspensions which are prepared by conventional methods may manifest variable, falsely low cholesterol solubility limits. We have developed two novel preparative methods which are specifically designed to prevent demixing during sample preparation. For detection of the cholesterol crystals, X-ray diffraction has proven to be quantitative and highly sensitive. Experiments based on these methods yield reproducible and precise cholesterol solubility limits: 66 mol% for phosphatidylcholine (PC) bilayers and 51 mol% for phosphatidylethanolamine (PE) bilayers. We present evidence that these are true, equilibrium values. In contrast to the dramatic headgroup effect (PC vs. PE), acyl chain variations had no effect on the cholesterol solubility limit in four different PC/cholesterol mixtures. (+info)
An ordered metastable phase in hydrated phosphatidylethanolamine: the Y-transition.
By using time-resolved X-ray diffraction, differential scanning calorimetry and scanning densitometry, we observed rapid formation at low temperature of a metastable ordered phase, termed LR1 phase, in fully hydrated dihexadecylphosphatidylethanolamine (DHPE). The LR1 phase has the same lamellar repeat period as the gel Lbeta phase but differs from the latter in its more ordered, orthorhombic hydrocarbon chain arrangement. It forms at about 12 degrees C upon cooling and manifests itself as splitting of the sharp, symmetric wide-angle X-ray peak of the DHPE gel phase into two reflections. This transition, designated the 'Y-transition', is readily reversible and proceeds with almost no hysteresis between cooling and heating scans. Calorimetrically, the LR1-->Lbeta transition is recorded as a low-enthalpy (0.2 kcal/mol) endothermic event. The formation of the LR1 phase from the gel phase is associated with a small, about 2 microl/g, decrease of the lipid partial specific volume recorded by scanning densitometry, in agreement with a volume calculation based on the X-ray data. The formation of the equilibrium Lc phase was found to take place from within the LR1 phase. This appears to be the only observable pathway for crystallisation of DHPE upon low-temperature incubation. Once formed, the Lc phase of this lipid converts directly into Lbeta phase at 50 degrees C, skipping the LR1 phase. Thus, the LR1 phase of DHPE can only be entered by cooling of the gel Lbeta phase. The data disclose certain similarities between the low-temperature polymorphism of DHPE and that of long-chain normal alkanes. (+info)
Ups and downs of protein crystallization: studies of protein crystals by high-performance capillary electrophoresis.
High-performance capillary electrophoresis is a high-technology micro-separation method. Short run time, full automation and minute amounts of sample make it a very attractive technique. In this report we describe studies of protein crystals by capillary electrophoresis. We show how high-performance capillary electrophoresis can be used effectively for rapid evaluation and examination of the protein solution used for crystallization, the protein crystals (solubilized) and surrounding mother liquor. With coated capillaries, the runs were reproducible and disturbing effects, such as electroendosmosis and interaction of the proteins with the capillary wall, were suppressed efficiently. We recommend this new technique as a powerful and routine companion to protein crystallography. (+info)
Protein folding at the air-water interface studied with x-ray reflectivity.
We report the results of x-ray reflectivity measurements of thin films formed by different water-soluble proteins at the air-aqueous solution interface. It is demonstrated that glucose oxidase, alcohol dehydrogenase, and urease molecules denaturate at the air-aqueous solution interface to form 8- to 14-A-thick peptide sheets. X-ray reflectivity data indicate that the spreading of a lipid monolayer at the aqueous solution surface before protein injection does not prevent proteins from unfolding. On the other hand, crosslinking of proteins results in intact enzyme layers at the subphase surface. A model that involves interaction of glucose oxidase molecules with a phospholipid monolayer is proposed. In this model, an observed decrease of the lipid electron density in the protein presence is explained in terms of "holes" in the monolayer film caused by protein molecule adsorption. (+info)
Structural characteristics of supramolecular assemblies formed by guanidinium-cholesterol reagents for gene transfection.
We have recently discovered that cationic cholesterol derivatives characterized by guanidinium polar headgroups are very efficient for gene transfection in vitro and in vivo. In spite of being based on some rationale at the molecular level, the development of these new synthetic vectors was nevertheless empirical. Indeed, the factors and processes underlying cationic lipid-mediated gene transfer are still poorly understood. Thus, to get a better insight into the mechanisms involved, we have examined the supramolecular structure of lipid/DNA aggregates obtained when using reagent bis(guanidinium)-tren-cholesterol (BGTC), either alone or as a liposomal formulation with the neutral phospholipid dioleoyl phosphatidylethanolamine (DOPE). We here report the results of cryotransmission electron microscopy studies and small-angle x-ray scattering experiments, indicating the presence of multilamellar domains with a regular spacing of 70 A and 68 A in BGTC/DOPE-DNA and BGTC-DNA aggregates, respectively. In addition, DNA lipoplexes with similar lamellar patterns were detected inside transfected HeLa cells by conventional transmission electron microscopy. These results suggest that DNA condensation by multivalent guanidinium-cholesterol cationic lipids involves the formation of highly ordered multilamellar domains, the DNA molecules being intercalated between the lipid bilayers. These results also invite further investigation of the intracellular fate of the internalized lipid/DNA structures during their trafficking toward the cell nucleus. The identification of the basic features of active complexes should indeed help in the design of improved guanidinium-based vectors. (+info)