D. L. D. Caspar

The Structure of Small Viruses

Publisher Summary This chapter deals with the structural studies on small ribonucleic acid (RNA)-containing viruses, and, in most detail, with tobacco mosaic virus (TMV). The TMV particle is characterized structurally by a small set of definite numbers giving the value of certain physically and chemically measurable quantities. The chapter considers those structural properties of TMV that can be accurately measured. X-ray studies have been made on a few of the small, stable, spherical viruses that contain as their major components only protein and ribonucleic acid. The relevant data on composition, dimensions, and particle weight are summarized, and the summary provides a comparison of values of the diameters obtained from the X-ray work with those found by electron microscopy. The structural information in these cases has largely come from electron microscopy, and these results are correlated with the structural principles that have emerged from the X-ray work. The investigations of substructure in both TMV and the crystalline spherical viruses, but particularly in the case of the former, have already made possible some generalizations about the way in which small viruses are put together.

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.

Tropomyosin: Crystal structure, polymorphism and molecular interactions☆

Abstract The α-protein tropomyosin forms a variety of ordered aggregates. True crystals with a very open lattice and at least two other net forms are produced near the isoelectric point together with fibrous aggregates. Three distinctive types of tactoids with axial periodicities about 400 A are formed with divalent cations. The electron microscope observations on the polymorphic forms have been related to X-ray diffraction measurements on the crystal lattice and the tactoids produced with magnesium. The X-ray pattern of one projection of the crystal has been interpreted at low resolution from a model composed of rod-shaped molecules arranged in accord with the electron micrographs. The tropomyosin molecules in the crystal are associated head-to-tail in polar filaments with a 400 A period. The filaments are periodically bent as a consequence of the cross connections at two sites alternatively separated by about 230 A and 170 A in the 400 A axial period. The principal conclusions of this study are: there is a specific polar end-to-end bonding of tropomyosin molecules which defines the period of about 400 A observed in the polymorphic nets and tactoids; there are two cross-connecting sites which are involved in net formation; in many of the forms, the polar filaments are arranged in oppositely directed pairs; the molecular coiled-coil is often supercoiled in the polar filaments. The periodicity in the I band of muscle can be identified with the 400 A repeat characteristic of the end-to-end association of tropomyosin. The polymorphism observed in vitro may be related to the structural and regulatory functions of tropomyosin in muscle.

Gap junction structures: V. Structural chemistry inferred from X-ray diffraction measurements on sucrose accessibility and trypsin susceptibility

X-ray diffraction patterns have been recorded from partially oriented specimens of gap junctions isolated from mouse liver and suspended in sucrose solutions of different concentration and thus of different electron density. Analysis of these diffraction patterns has shown that sucrose is excluded from the 6-fold rotation axis of the junction lattice for a length of about 100 A. This indicates that the aqueous channel of the junctions is in the closed, high resistance state in these preparations. Mapping of the sucrose-accessible space in the junction indicates that the cross-sectional area of the channel entrance on the cytoplasmic side of the membrane could be up to five times larger than the area of the transmembrane channel. Sucrose does not penetrate more than 20 A into the membrane along the channel. Apparently the aqueous channel, 8 to 10 A in radius for most of its length, is narrowed or blocked by a small feature about 50 A from the center of the gap. Very close interactions exist between the gap junction protein and the lipid polar head groups on the cytoplasmic surface of the membrane. In this region, the protein intercalates between the polar head groups. These results suggest that the gap junction protein may have a functional two-domain structure. One domain, with a molecular weight of about 15,000, spans one bilayer and half of the gap and is contained largely within a radius of 25 A from the 6-fold axis. The second domain is smaller and occupies the cytoplasmic surface of the gap junction membrane. Trypsin digestion removes about 4000 Mr from the cytoplasmic surface domain of the junction protein. Most of the material susceptible to trypsin digestion is located more than 28 A from the 6-fold axis.

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.

Diffraction Studies of Molecular Organization and Membrane Interactions in Myelin

The myelin sheath derives from the spiral infolding about the axon of a membrane-bound, glial cell process. The major part of the sheath consists of closely packed membrane pairs separated by narrow fluid spaces. The periodic nature of this membrane array makes myelin well suited for examination of its molecular organization by diffraction techniques. Diffraction provides a means of monitoring the separation between membranes and of analyzing the forces and interactions between them. This method, which is nonperturbing, is uniquely suited to analyzing myelin structure and stability in physiologically intact tissue, even in the living animal. X-ray and neutron diffraction results on myelin can be correlated with its chemical composition, its structure as seen by electron microscopy (EM), and its responses to nerve conduction and to physical-chemical treatments. This correlation has led to a description of the average distribution of lipid, protein, and water in the membrane array and to the localization of specific proteins and lipids within the myelin membrane bilayer. It has also led to an understanding of the role of ions in membrane-membrane interactions in myelin and the possible involvement of these ions in nerve conduction.

Cross-beta Order and Diversity in Nanocrystals of an Amyloid-forming Peptide

The seven-residue peptide GNNQQNY from the N-terminal region of the yeast prion protein Sup35, which forms amyloid fibers, colloidal aggregates and highly ordered nanocrystals, provides a model system for characterizing the elusively protean cross-beta conformation. Depending on preparative conditions, orthorhombic and monoclinic crystals with similar lath-shaped morphology have been obtained. Ultra high-resolution (<0.5A spacing) electron diffraction patterns from single nanocrystals show that the peptide chains pack in parallel cross-beta columns with approximately 4.86A axial spacing. Mosaic striations 20-50 nm wide observed by electron microscopy indicate lateral size-limiting crystal growth related to amyloid fiber formation. Frequently obtained orthorhombic forms, with apparent space group symmetry P2(1)2(1)2(1), have cell dimensions ranging from /a/=22.7-21.2A, /b/=39.9-39.3A, /c/=4.89-4.86A for wet to dried states. Electron diffraction data from single nanocrystals, recorded in tilt series of still frames, have been mapped in reciprocal space. However, reliable integrated intensities cannot be obtained from these series, and dynamical electron diffraction effects present problems in data analysis. The diversity of ordered structures formed under similar conditions has made it difficult to obtain reproducible X-ray diffraction data from powder specimens; and overlapping Bragg reflections in the powder patterns preclude separated structure factor measurements for these data. Model protofilaments, consisting of tightly paired, half-staggered beta strands related by a screw axis, can be fit in the crystal lattices, but model refinement will require accurate structure factor measurements. Nearly anhydrous packing of this hydrophilic peptide can account for the insolubility of the crystals, since the activation energy for rehydration may be extremely high. Water-excluding packing of paired cross-beta peptide segments in thin protofilaments may be characteristic of the wide variety of anomalously stable amyloid aggregates.

The symmetries of filamentous phage particles.

Abstract The helical symmetries of two classes of filamentous bacteriophage particles are distinctly different. The symmetry of the class I particles is † C 5 S ~2.0 (a 5-fold rotation axis combined with an approximately 2-fold screw axis). The symmetry of the class II particles is C 1 S 5.4 (a one-start helix with 27 subunits equally spaced along five turns). The same basic α-helical interlocking arrangement of the largely α-helical coat protein subunits can be accommodated by the symmetry of the two classes of phage particles. The conservation of this structural pattern reflects intrinsic packing properties of α-helices. The difference between the symmetries of the class I and class II particles suggests that different assembly processes may have evolved to form these structures with very similar protein packing architectures.

Switching in the self-assembly of tobacco mosaic virus

Experimental observations on the structure and physicochemical properties of TMV protein assemblies have led to a fundamental switch in the model of the self-assembly process: rather than being nucleated by the hypothetical two-layer disk, virus assembly appears to be initiated by interaction of the specific RNA sequence with a short helical aggregate of the coat protein arranged as in the virus. Formation of the 20s nucleating aggregate involves the binding of an average of half a proton per protein subunit. This proton-binding site can be identified with the carboxyl-carboxylate pair that is formed between top and bottom protein surfaces at a radius of 58 A in the virus helix. Because the 20s aggregate consists of about two helical turns, only one carboxyl-carboxylate pair will be formed between each top-bottom pair of protein subunits. Limitation of the length of the 20s helical aggregate at neutral pH can be accounted for by disorder of the inner loop of the protein chain, due to electrostatic repulsion among the carboxyl groups that form the anomalous proton-binding site at 25 A radius in the ordered virus structure. To grow beyond two to three turns, inner loops of the protein at the interior of the helix must be ordered in the close-packed arrangement. The electrostatic repulsion opposing this ordering can be overcome by binding of the viral RNA at neutral pH, by calcium binding, or by proton binding in slightly acid solution. Virus disassembly upon infection appears to result from the low intracellular calcium and proton concentration compared to the extracellular environment, which increases the electrostatic repulsion among the negatively charged groups involved in calcium and proton binding, thereby allowing cellular ribosomes to competitively bind the viral RNA. Disk aggregates of TMV protein, which form at high ionic strength in alkaline solution, do not appear to be involved in virus assembly. The stacked-disc aggregate, which was previously presumed to be built of a polar stack of the hypothetical polar two-layer aggregate, is, in fact, a bipolar structure. Because the bonding between turns of the disc structures is different from that of the virus helix, direct switching between these structures by the postulated dislocation does not occur. TMV assembly does appear to involve conservation of bonding specificity, as initially presumed, but only in helical packing arrangements of the protein subunits. Switching from disordered to ordered conformations of the protein, dependent on changes in the electrostatic interactions among the protein subunits, appears to be critical in controlling the assembly process.

Refined Atomic Model of the Four-Layer Aggregate of the Tobacco Mosaic Virus Coat Protein at 2.4-Å Resolution

Previous x-ray studies (2.8-A resolution) on crystals of tobacco mosaic virus coat protein grown from solutions containing high salt have characterized the structure of the protein aggregate as a dimer of a bilayered cylindrical disk formed by 34 chemically identical subunits. We have determined the crystal structure of the disk aggregate at 2.4-A resolution using x-ray diffraction from crystals maintained at cryogenic temperatures. Two regions of interest have been extensively refined. First, residues of the low-radius loop region, which were not modeled previously, have been traced completely in our electron density maps. Similar to the structure observed in the virus, the right radial helix in each protomer ends around residue 87, after which the protein chain forms an extended chain that extends to the left radial helix. The left radial helix appears as a long alpha-helix with high temperature factors for the main-chain atoms in the inner portion. The side-chain atoms in this region (residues 90-110) are not visible in the electron density maps and are assumed to be disordered. Second, interactions between subunits in the symmetry-related central A pair have been determined. No direct protein-protein interactions are observed in the major overlap region between these subunits; all interactions are mediated by two layers of ordered solvent molecules. The current structure emphasizes the importance of water in biological macromolecular assemblies.