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Biomedical subjects

G Stubbs

Publications and source records attributed to G Stubbs.

At least 19 recordsLinked to original sources
PubMed1996

Carboxylate interactions involved in the disassembly of tobacco mosaic tobamovirus.

Structural studies of tobacco mosaic tobamovirus (TMV) have identified two coat protein (CP) intersubunit carboxyl-carboxylate interactions and one CP carboxylate-RNA phosphate interaction whose electrostatic repulsion is believed to drive virion disassembly. In this study, the involvement of each interaction in the disassembly process was examined. Site-directed mutagenesis was used to replace selected negatively charged CP residues, E or D, with neutral residues, Q or N, respectively. Purified mutant CPs were assayed for their ability to inhibit wild-type TMV disassembly both in vitro and in vivo. Results indicate that the lateral carboxylate interaction made by residue E106 is much more complex than previously thought, involving three residues, E95, E97, and D109, from an adjacent subunit. Mutations at all three residues are required to inhibit disassembly significantly. Different mutant coat proteins inhibited disassembly of the wild-type virus to varying degrees. Mutant E50Q, which modified the axial intersubunit interaction, had the greatest ability to inhibit disassembly followed by mutants E95Q/E97Q/D109N and D116N, which modified the lateral and CP-RNA interactions, respectively. Within each set of interacting carboxylate groups, mutations in the face opposite the disassembling surface of the TMV virion conferred the greatest ability to inhibit disassembly. This observation is consistent with the polar nature of TMV disassembly and confirms that repulsive intersubunit interactions derived from the 5' terminal subunits provide the key controlling mechanisms for virion disassembly.

Capsid↗
PubMed1995

Site-directed mutagenesis confirms the involvement of carboxylate groups in the disassembly of tobacco mosaic virus.

Electrostatic repulsion between carboxylate groups across subunit interfaces has for many years been recognized as important in the disassembly of simple plant viruses. In the coat protein of tobacco mosaic virus (TMV), the amino acids Glu50 and Asp77 have been proposed as examples of such carboxylate groups. Site-directed mutagenesis has been used to replace these amino acids by Gln and Asn, respectively. Increased virion stability, together with reduced infectivity and reduced capacity for long-distance transport within the host plant confirms that the negative charges on the side chains of these amino acids are involved in the disassembly of TMV. Mixing purified mutant coat proteins with wild-type virions under appropriate conditions stabilizes the virions to alkaline disassembly and reduces their infectivity. It is suggested that transgenic plants expressing such mutant coat proteins could have enhanced resistance to virus infection.

Capsid↗
PubMed1994

Structure-function relationship between tobacco mosaic virus coat protein and hypersensitivity in Nicotiana sylvestris.

Alterations in the structure of the tobacco mosaic virus (TMV) coat protein affect the elicitation of the N' gene hypersensitive response (HR) in Nicotiana sylvestris. To investigate this structure-function relationship, amino acid substitutions with predicted structural effects were created throughout the known structure of the TMV coat protein. Substitutions that resulted in the elicitation of the HR resided within and would predictably interfere with interface regions located between adjacent subunits in ordered aggregates of coat protein. Substitutions that did not result in the elicitation of the HR were either conservative or located outside these interface regions. In vitro analysis of coat protein aggregates demonstrated HR-eliciting coat proteins to have reduced aggregate stability in comparison with non-HR-eliciting coat proteins and a correlation existed between the strength of the elicited HR and the ability of a substitution to interfere with ordered aggregate formation. This finding corresponded with the predicted structural effects of HR-eliciting substitutions. Radical substitutions that predictably disrupted coat protein tertiary structure were found to prevent HR elicitation. These findings demonstrate that structural alterations that affect the stability of coat protein quaternary structure but not tertiary structure lead to host cell recognition and HR elicitation. A model for HR elicitation is proposed, in which disassembly of coat protein aggregates exposes a host "receptor" binding site.

Amino Acids↗
PubMed1994

Structure determination of cucumber green mottle mosaic virus by X-ray fiber diffraction. Significance for the evolution of tobamoviruses.

Cucumber green mottle mosaic virus (CGMMV) is a rod-shaped virus of the tobacco mosaic virus (TMV) group. The structure of cucumber green mottle mosaic virus has been determined by fiber diffraction methods at 3.4 A resolution, and refined by molecular dynamics methods to an R factor of 0.093. Disassembly of TMV is driven by the mutual repulsion of intersubunit carboxyl-carboxylate pairs, but one of these pairs is not conserved in CGMMV. An alternative pair, located about 5 A from the site of the TMV pair, has been found in CGMMV. Comparison of the two structures suggests that the carboxylate groups are free to migrate in the subunit interfaces during evolution.

Biological Evolution↗
PubMed1993

Crystallization and preliminary X-ray analysis of papaya mosaic virus coat protein.

Papaya mosaic virus coat protein has been treated with trypsin and a large fragment of the intact protein has been crystallized in space group P3(1)21 or P3(2)21 (unit cell dimensions: a = b = 110 A, c = 237 A). The crystals diffract to 3.5 A resolution. Crystals of the untreated protein have also been grown. The untreated protein crystals diffract to 4 A resolution, but have a large mosaic spread. They have the same space group as the trypsin-treated protein crystals, but a much smaller unit cell (a = b = 72 A, c = 240 A).

Capsid↗
PubMed1993

Preliminary X-ray diffraction studies of ribgrass mosaic virus.

Fiber diffraction data were collected from oriented sols of ribgrass mosaic virus and a lead derivative of the virus. Two lead binding sites were found. Two intersubunit carboxylcarboxylate pairs, different from those in other tobamoviruses, are predicted to control viral assembly and disassembly. One of the carboxyl-carboxylate pairs forms part of a lead binding site.

Amino Acid Sequence↗
PubMed1993

Molecular dynamics in refinement against fiber diffraction data.

The molecular dynamics (MD) method has been adapted for refinement of the structures of helical macromolecular aggregates aginst X-ray fiber diffraction data. To test the effectiveness of the method, refinements of the tobacco mosaic virus structure were carried out against a set of simulated fiber diffraction intensities using the MD method as well as the conventional restrained least-squares (RLS) method. The MD refinement converged to a very low R factor and produced a structure with generally satisfactory sterochemistry, while the RLS refinemnt was trapped at a local energy minimum with a larger R factor. Results suggest that the effective experimental radius of convergence of the MD method is significantly greater than that of the RLS method. Even when the initial structure is too far from the true structure to allow direct refinement, the MD method is able to find local minima that resemble the true structure sufficiently to allow improved phasing and thus lead to interpretable difference maps for model rebuilding.

Capsid↗
PubMed1992

Structure of the U2 strain of tobacco mosaic virus refined at 3.5 A resolution using X-ray fiber diffraction.

The structure of the U2 strain of tobacco mosaic virus (TMV) has been determined by fiber diffraction methods at 3.5 A resolution, and refined by a combination of restrained least-squares and molecular dynamics methods to an R-factor of 0.096. The structure is extremely similar to that of the common strain of TMV, with the largest differences being in the protein loop that makes up the inner surface of the virus, and in the C-terminal region on the outer surface. Differences in the inner loop can be correlated with differences in the properties of the two viruses.

Capsid↗
PubMed1992

Characterization of a putative calcium-binding site in tobacco mosaic virus.

Lead has been used as a substitute for calcium binding to tobacco mosaic virus (TMV). The high atomic number of lead has allowed us to use difference maps from X-ray fiber diffraction data to characterize a calcium-binding site in the virus. The metal ligands are slightly different from those previously believed to bind calcium to TMV, although the binding site is very close to one previously described. Two acetate groups are also bound to the lead atom. There is no significant backbone conformational change in the protein as a result of metal binding; the binding is accomplished by means of relatively small movements in amino acid side chains.

Binding Sites↗
PubMed1990

Fiber diffraction analysis of cucumber green mottle mosaic virus using limited numbers of heavy-atom derivatives.

The structure of cucumber green mottle mosaic virus has been determined from fiber diffraction data to a resolution of 5 A, using only two derivatives and without making any specific assumptions about the molecular structure of the virus. Because of the cylindrical averaging of fiber diffraction data, large numbers of heavy-atom derivatives have been required in previous structure determinations, but it is shown that simplifying assumptions about the overlapping intensities in fiber diffraction are sufficient for structure determination at this resolution using only two derivatives.

Image Processing, Computer-Assisted↗
PubMed1990

Preliminary crystallographic examination of a novel fungal lysozyme from Chalaropsis.

The lysozyme from the fungus of the Chalaropsis species has been crystallized. This lysozyme (Mr 22,415) displays no sequence homology with avian, phage, or mammalian lysozymes, however, preliminary studies indicate significant sequence homology with the bacterial lysozyme from Streptomyces. Both enzymes are unusual in possessing beta-1,4-N-acetylmuramidase and beta-1,4-N,6-O-diacetylmuramidase activity. The crystals grow from solutions of ammonium sulfate during growth periods from several months to a year. The space group is P2(1)2(1)2(1) with a = 34.0 A, b = 42.6 A, c = 122.1 A. Preliminary data indicate that there is 1 molecule/asymmetric unit. A complete native data set has been collected to 2.57-A resolution. The crystals are highly ordered and exhibit diffraction patterns to d-spacings less than 1.5 A.

Crystallization↗
PubMed1989

Visualization of protein-nucleic acid interactions in a virus. Refined structure of intact tobacco mosaic virus at 2.9 A resolution by X-ray fiber diffraction.

The structure of tobacco mosaic virus (TMV) has been determined by fiber diffraction methods at 2.9 A resolution, and refined by restrained least-squares to an R-factor of 0.096. Protein-nucleic acid interactions are clearly visible. The final model contains all of the non-hydrogen atoms of the RNA and the protein, 71 water molecules, and two calcium-binding sites. Viral disassembly is driven by electrostatic repulsions between the charges in two carboxyl-carboxylate pairs and a phosphate-carboxylate pair. The phosphate-carboxylate pair and at least one of the carboxyl-carboxylate pairs appear to be calcium-binding sites. Nucleotide specificity, enabling TMV to recognize its own RNA by a repeating pattern of guanine residues, is provided by two guanine-specific hydrogen bonds in one of the three base-binding sites.

Amino Acids↗
PubMed1989

The probability distributions of X-ray intensities in fiber diffraction: largest likely values for fiber diffraction R factors.

R factors in fiber diffraction are generally lower than in conventional crystallography, because of the cylindrical averaging of fiber diffraction data. The probability distributions for fiber diffraction intensities, analogous to Wilson's distributions for crystal diffraction intensities, are derived, and from these the largest likely values of R are estimated. These values depend on the size and symmetry of the diffracting particle and on the resolution of the analysis, and range from 0.586 for systems for very high symmetry (as in crystal diffraction) to much lower values for systems of low symmetry.

Crystallography↗
PubMed1989

Visualization of alpha-helices in tobacco mosaic virus by cryo-electron microscopy.

We have used tobacco mosaic virus (TMV) as a test specimen, in order to develop techniques for the analysis of high-resolution structural detail in electron micrographs of biological assemblies with helical symmetry. It has previously been shown that internal details of protein structure can be visualized by processing electron micrographs of unstained specimens of extended two-dimensional crystalline arrays. However, the techniques should in principle be applicable to other periodic specimens, such as assemblies with helical symmetry. We show here that data to spacings better than 10 A can be retrieved from electron images of frozen hydrated TMV. The three-dimensional computed map agrees well with that derived from X-ray diffraction and shows the two pairs of alpha-helices forming the core of the coat subunit, the C alpha-helix and the viral RNA. The results demonstrate that it is possible to determine detailed internal structure in helical particles.

Freezing↗
PubMed1988

Enhancement and simplification of macromolecular images.

Computer graphics programs have been devised to display selected atomic features and to simplify images of complex macromolecular structures. By using boundary outlines, adjustment of size and shape of the molecular components, color coding, shading, and selective omission of obscuring detail, attention can be focused on specific interactions which determine higher levels of organization. A balanced color table has been constructed in which different hues have equal steps in brightness; this table has facilitated distinction of atom types and sequence coding together with representation of an optimum range of depth cueing and surface shading. The graphics system has been used with the atomic coordinates of the tobacco mosaic virus structure to simplify images of the protein subunit, to illustrate intermolecular interactions, and to relate subunit packing arrangements in different assemblies to the underlying atomic structure. The system has also been used to construct a schematic representation of the polyomavirus capsid, based on low resolution data. Application of artistic methods contributes to the effective presentation and interpretation of detailed scientific information about complex macromolecular structures.

Computer Graphics↗
PubMed1987

Structure of microtubules with reduced hydration. Comparison of results from X-ray diffraction and electron microscopy.

A recent model for the structure of microtubules is used to interpret X-ray fiber diffraction patterns from microtubules, obtained under various conditions. The results suggest that tubulin may undergo conformational changes under conditions of reduced water-activity. Such changes could account for some of the differences in the structure of tubulin as determined by electron microscopy and X-ray diffraction.

Microscopy, Electron↗