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D M Salunke

Publications and source records attributed to D M Salunke.

8 recordsLinked to original sources
PubMed1989

Polymorphism in the assembly of polyomavirus capsid protein VP1.

Polyomavirus major capsid protein VP1, purified after expression of the recombinant gene in Escherichia coli, forms stable pentamers in low-ionic strength, neutral, or alkaline solutions. Electron microscopy showed that the pentamers, which correspond to viral capsomeres, can be self-assembled into a variety of polymorphic aggregates by lowering the pH, adding calcium, or raising the ionic strength. Some of the aggregates resembled the 500-A-diameter virus capsid, whereas other considerably larger or smaller capsids were also produced. The particular structures formed on transition to an environment favoring assembly depended on the pathway of the solvent changes as well as on the final conditions. Mass measurements from cryoelectron micrographs and image analysis of negatively stained specimens established that a distinctive 320-A-diameter particle consists of 24 close-packed pentamers arranged with octahedral symmetry. Comparison of this unexpected octahedral assembly with a 12-capsomere icosahedral aggregate and the 72-capsomere icosahedral virus capsid by computer graphics methods indicates that similar connections are made among trimers of pentamers in these shells of different size. The polymorphism in the assembly of VP1 pentamers can be related to the switching in bonding specificity required to build the virus capsid.

Capsid↗
PubMed1988

Liquid-like movements in crystalline insulin.

Diffuse X-ray scattering from protein crystals provides information about molecular flexibility and packing irregularities. Here we analyse diffraction patterns from insulin crystals that show two types of scattering related to disorder: very diffuse, liquid-like diffraction, and haloes around the Bragg reflections. The haloes are due to coupled displacements of neighbouring molecules in the lattice, and the very diffuse scattering results from variations in atomic positions that are only locally correlated within each molecule. The measured intensity was digitally separated into three components: the Bragg reflections and associated haloes; the water and Compton scattering; and the scattering attributed to internal protein movements. We extend methods used to analyse disorder in membrane structures to simulate the diffuse scattering from crystalline insulin in terms of (1) the Patterson (autocorrelation) function of the ideal, ordered crystal structure, (2) the root-mean-square (r.m.s.) amplitude of the atomic movements, and (3) the mean distance over which these displacements are coupled. Movements of the atoms within the molecules, with r.m.s. amplitudes of 0.4-0.45 A, appear to be coupled over a range of approximately 6 A, as in a liquid. These locally coupled movements account for most of the disorder in the crystal. Also, the protein molecules, as a whole, jiggle in the lattice with r.m.s. amplitudes of approximately 0.25 A that appear to be significantly correlated only between nearest neighbours.

Crystallization↗
PubMed1986

Disk aggregates of tobacco mosaic virus protein in solution: electron microscopy observations.

Previous studies of the coat protein of tobacco mosaic virus (TMVP) have shown that TMVP presumably exists as linear stacks of two-ring cylindrical disks in the 0.7 M ionic strength buffer used for crystallizing the disks for X-ray diffraction studies [Raghavendra, K., Adams, M.L., & Schuster, T.M. (1985) Biochemistry 24, 3298-3304]. The spectroscopic and sedimentation studies of solutions of TMVP under these crystallizing conditions have demonstrated a long-term metastability of these disk aggregates when they are placed in 0.1 M ionic strength buffers, as are used for reconstituting tobacco mosaic virus from TMVP and viral RNA. The present work describes an electron microscopic study of TMVP disk aggregates under the same solution conditions employed in the previous spectroscopic and sedimentation studies. The results show that in the pH 8.0 0.7 M ionic strength crystallization buffer TMVP exists as stacks of disks which range in size from about 6 to 24 layers, corresponding to 3-12 2-layer disk aggregates having 17 subunits per layer. These TMVP aggregates persist in a metastable form in 0.1 M ionic strength virus reconstitution buffer with no apparent changes in structure of the stacked disks. The results are consistent with the conclusions of the solution physical-chemical studies which suggest that the disk structure may not be related to the 20S TMVP aggregate that is the nucleation species in virus

Crystallization↗
PubMed1986

Self-assembly of purified polyomavirus capsid protein VP1.

The polyomavirus major capsid protein VP1, purified after expression of the recombinant gene in E. coli, was isolated as oligomers resembling the dissociated capsomeres derived from viral capsids. Image analysis of low-dose electron micrographs demonstrates that these VP1 oligomers are exclusively pentamers. The purified VP1 pentamers associated to form capsid-like assemblies and polymorphic aggregates at high ionic strength. The capsid-like assemblies were stabilized at low ionic strength by the addition of calcium. Self-assembly of the unmodified, recombinant DNA-generated VP1 implies that the posttranslational charge modifications of VP1 and the minor virion protein components, VP2 and VP3, are not essential for capsid formation. The nonequivalently related subunits of the penta- and hexavalent capsomeres therefore must spontaneously switch their bonding specificity during assembly.

Capsid↗
PubMed1985

Arrangement of subunits in peanut lectin. Rotation function and chemical cross-linking studies.

X-ray intensity data from the native orthorhombic crystals of peanut lectin have been collected using oscillation photography. Rotation function studies using data up to a resolution of 4.5 A indicate that the four subunits in the molecule, which constitute the asymmetric unit in the crystals, are related to one another by three mutually perpendicular noncrystallographic 2-fold axes. Chemical cross-linking experiments in solution followed by sodium dodecyl sulfate gel electrophoresis, carried out in parallel, suggest that there is more than one type of intersubunit approach in the molecule. Rotation function and cross-linking studies thus show that the tetrameric molecule of peanut lectin is a dimer of a dimer. The two monomers in a dimer are related by a 2-fold axis. The two dimers are in turn related by another 2-fold axis perpendicular to the one that relates the two monomers in the dimer, endowing the molecule with 222 (D2) symmetry.

Cross-Linking Reagents↗
PubMed1985

X-ray characterisation of an additional binding site in lysozyme.

Bromophenol red (BPR) binds to lysozyme and inhibits its activity against bacterial cell walls, but not against the polysaccharide component of peptidoglycan. The binding site of BPR in the enzyme has been characterised by X-ray analysis of the complex at 5.5A resolution. The new binding site, which is outside the cleft close to subsite F, is presumably involved in interactions with the peptide component of peptidoglycan, in the action of lysozyme against bacterial cell walls.

Animals↗
PubMedDate not supplied

Site-directed mutation affecting polyomavirus capsid self-assembly in vitro.

Nonequivalent bonding of identical protein subunits occurs in the polyomavirus capsid were identical pentameric capsomeres occupy both hexavalent and pentavalent positions in the icosahedral surface lattice. The polyomavirus major capsid protein VP1, purified after expression of the recombinant gene in Escherichia coli, has been isolated as capsomeres that self-assemble into capsid-like structures in vitro. The ability to switch bonding specificity in different symmetry environments therefore must be intrinsic to the VP1 molecule. In vitro self-assembly provides an assay for VP1 mutations affecting capsomere and capsid formation. We report here that a directed mutation in the VP1 expression vector, leading to a protein truncated at the carboxy terminus, results in a mutant VP1 that forms capsomeres, but not capsids, in the in vitro assembly assay. The carboxy terminus of VP1 therefore appears to be involved in the specific bonding responsible for the non-equivalent association of capsomeres.

Amino Acid Sequence↗