Disassembly Of Particles Research Articles

In vitro disassembly of native and reconstituted hybrid particles of cowpea strain tobacco mosaic virus

In vitro disassembly of native and reconstituted hybrid particles of cowpea strain tobacco mosaic virus

Rearrangement of nucleosomal components by modification of histone amino groups. Structural role of lysine residues.

Modification of nucleosomal particles from chicken erythrocytes with the reagents for protein amino groups acetic and dimethylmaleic anhydrides causes a rearrangement of nucleosomal components. Treatment with both reagents is accompanied by liberation of free DNA and formation of residual particles with anomalous histone composition. The residual particles obtained with acetic anhydride contain an excess of histones corresponding to the free DNA produced. In contrast, dimethylmaleic anhydride causes release of histones H1, H5, H2A and H2B and formation of residual particles deficient in these histones but containing an excess of H3 and H4 corresponding to the liberated DNA. Regeneration of the modified amino groups of nucleosomal preparations treated with dimethylmaleic anhydride is accompanied by reconstitution of nucleosomal particles with the sedimentation coefficient and composition of core histones of the original nucleosomes. This reconstitution does not occur when the released fraction containing histones H2A and H2B and free DNA is separated from the residual particles. The studied disassembly of nucleosomal particles obtained by specifically blocking lysine-DNA interactions with these reagents appears to indicate that lysine residues are essential for the binding of DNA to histones with formation of nucleosomal particles.

Establishment and analysis of a system which allows assembly and disassembly of alphavirus core-like particles under physiological conditions in vitro

Core-like (CL) particles which closely resemble alphavirus cores in size, shape, and relative amount of nucleic acid and protein have been assembled in vitro from Sindbis (SIN) virus core (C) protein and single-stranded nucleic acids in buffer containing 1 M urea [ G. Wengler, U. Boege, G. Wengler, H. Bischoff, and K. Wahn (1982) Virology 118, 401–410]. We have now analyzed the interaction of SIN virus C protein and nucleic acids in vitro under conditions designed to resemble those present in the cell during core assembly. In buffer containing 100 m M K-acetate, 1.7 m M Mg-acetate, pH 7.4, CL particles are efficiently assembled from all single-stranded nucleic acids analyzed, and even heparin and polyvinylsulfate are incorporated into such particles. A reticulocyte lysate translates SIN virus-specific mRNA into C protein under these ionic conditions. Interactions of C protein with nucleic acids and ribosomes in a reticulocyte lysate have also been analyzed. The following conclusions can be drawn from these analyses: (1) In accordance with earlier findings [ N. Glanville and I. Ulmanen (1976) Biochem. Biophys. Res. Commun. 71, 393–399] the C protein translated in vitro efficiently binds to ribosomes. (2) Exogenously added C protein binds to the large subunit of the ribosomes in the lysate. (3) CL particles can be assembled in the lysate from exogenous added 42 S genome RNA and exogenous added C protein if both components are present at sufficiently high concentrations. (4) The C protein translated from viral mRNA in the lysate is transferred from the ribosomes into preassembled CL particles containing 42 S RNA in the lysate. (5) If only small amounts of CL particles are added into a lysate these particles disaggregate and core protein molecules are transferred from the particles to the large subunit of the ribosomes. The results on the assembly of CL particles in vitro allow the formulation of some hypotheses concerning the assembly and disassembly of core particles in vivo.

Microtubule nucleation sequence studied by time-resolved x-ray scattering and electron microscopy

When a solution of microtubule protein is changed from non-polymerising to polymerising conditions (e.g. by temperature jump or mixing with GTP) there is a series of structural transitions preceding microtubule growth. These have been detected by time-resolved X-ray scattering using synchrotron radiation, and they may be classified into pre-nucleation and nucleation events. X-ray patterns are good indicators for the average behavior of the particles in solution, but they are difficult to interpret unless additional information on their structure is available. We therefore studied the assembly process by electron microscopy under conditions approaching those of the X-ray experiment. There are two difficulties in the EM approach: One is that the particles important for assembly are usually small and not very regular and therefore tend to be overlooked. Secondly EM specimens require low concentrations which favor disassembly of the particles one wants to observe since there is a dynamic equilibrium between polymers and subunits.

Cooperative exposure of histone H3 thiols in core particles

The kinetics of thiol modification of histone H3 from chicken erythrocyte nuclei with the fluorogenic compound N-[p-(2-benzimidazolyl)phenyl]maleimide was determined at pH 5.5 and 2°C. Comparative experiments were performed with H3 in the natural mixture of the other histones (whole histone), assembled in the nucleosomal and in H2a/H2b depleted core particles. Exposure of the H3 thiols in core particles occurs within a rather narrow NaCl concentration range. The transition midpoint is shifted to lower ionic strength with decreasing core particle concentration and with increasing concentration of ethidium bromide added. The results presented permit the following conclusions about the disassembly process of core particles. Release of the H2a/H2b pairs at 0.5–0.7 m NaCl is not directly correlated with an exposure of H3 thiols. Exposure occurs at around 0.1 m NaCl and starts with a rather fast conformational transition of the depleted core followed by histone-DNA dissociation. Comparative experiments exploring the air mediated oxidation of thiols and the reactivity of lysine side chains with fluorescamine illustrate that the thiol exposure is a rather distinct event in core particle disassembly.