Phase diagram of self-assembled viral capsid protein polymorphs.

We present an experimental study of the self-assembly of capsid proteins of the cowpea chlorotic mosaic virus (CCMV), in the absence of the viral genome, as a function of pH and ionic strength. In accord with previous measurements, a wide range of polymorphs can be identified by electron microscopy, among them single and multiwalled shells and tubes. The images are analyzed with respect to size and shape of aggregates, and evidence is given that equilibrium has been achieved, allowing a phase diagram to be constructed. Some previously unreported structures are also described. The range and stability of the polymorphs can be understood in terms of electrostatic interactions and the way they affect the spontaneous curvature of protein networks and the relative stabilities of pentamers and hexamers.

Polymerization of the SAM domain of TEL in leukemogenesis and transcriptional repression.

TEL is a transcriptional repressor that is a frequent target of chromosomal translocations in a large number of hematalogical malignancies. These rearrangements fuse a potent oligomerization module, the SAM domain of TEL, to a variety of tyrosine kinases or transcriptional regulatory proteins. The self-associating property of TEL-SAM is essential for cell transformation in many, if not all of these diseases. Here we show that the TEL-SAM domain forms a helical, head-to-tail polymeric structure held together by strong intermolecular contacts, providing the first clear demonstration that SAM domains can polymerize. Our results also suggest a mechanism by which SAM domains could mediate the spreading of transcriptional repression complexes along the chromosome.

Identification of a subunit interface in transthyretin amyloid fibrils: evidence for self-assembly from oligomeric building blocks.

Amyloid and prion diseases appear to stem from the conversion of normally folded proteins into insoluble, fiber-like assemblies. Despite numerous structural studies, a detailed molecular characterization of amyloid fibrils remains elusive. In particular, models of amyloid fibrils proposed thus far have not adequately defined the constituent protein subunit interactions. To further our understanding of amyloid structure, we employed thiol-specific cross-linking and site-directed spin labeling to identify specific protein-protein associations in transthyretin (TTR) amyloid fibrils. We find that certain cysteine mutants of TTR, when dimerized by chemical cross-linkers, still form fibers under typical in vitro fibrillogenic conditions. In addition, site-directed spin labeling of many residues at the natural dimer interface reveals that their spatial proximity is preserved in the fibrillar state even in the absence of cross-linking constraints. Here, we present the first view of a subunit interface in TTR fibers and show that it is very similar to one of the natural dimeric interchain associations evident in the structure of soluble TTR. The results clarify varied models of amyloidogenesis by demonstrating that transthyretin amyloid fibrils may assemble from oligomeric protein building blocks rather than structurally rearranged monomers.

Design of three-dimensional domain-swapped dimers and fibrous oligomers.

Three-dimensional (3D) domain-swapped proteins are intermolecularly folded analogs of monomeric proteins; both are stabilized by the identical interactions, but the individual domains interact intramolecularly in monomeric proteins, whereas they form intermolecular interactions in 3D domain-swapped structures. The structures and conditions of formation of several domain-swapped dimers and trimers are known, but the formation of higher order 3D domain-swapped oligomers has been less thoroughly studied. Here we contrast the structural consequences of domain swapping from two designed three-helix bundles: one with an up-down-up topology, and the other with an up-down-down topology. The up-down-up topology gives rise to a domain-swapped dimer whose structure has been determined to 1.5 A resolution by x-ray crystallography. In contrast, the domain-swapped protein with an up-down-down topology forms fibrils as shown by electron microscopy and dynamic light scattering. This demonstrates that design principles can predict the oligomeric state of 3D domain-swapped molecules, which should aid in the design of domain-swapped proteins and biomaterials.

The structure of isometric capsids of bacteriophage T4.

The three-dimensional structure of DNA-filled, bacteriophage T4 isometric capsids has been determined by means of cryoelectron microscopy and image reconstruction techniques. The packing geometry of protein subunits on the capsid surface was confirmed to be that of the triangulation class T = 13. The reconstruction clearly shows pentamers, attributed to capsid protein gp24*, surrounded by hexamers of the major capsid protein, gp23*. Positions of the accessory proteins, Hoc and Soc, are also clearly delineated in the surface lattice. The Hoc protein is the most prominent surface feature and appears as an extended molecule with a rounded base from which a thin neck and a globular head protrude. One Hoc molecule associates with each hexamer. Nearly continuous "ridges" are formed at the periphery of the gp23* hexamers by an association of 12 Soc molecules; however, Soc is absent along the boundaries between the hexamers and the pentamers. The duplex DNA genome forms a highly condensed series of concentric layers, spaced about 2.36 nm apart, that follow the general contour of the inner wall of the protein capsid.

Bacteriophage T4 self-assembly: localization of gp3 and its role in determining tail length.

Gene 3 of bacteriophage T4 participates at a late stage in the T4 tail assembly pathway, but the hypothetical protein product, gp3, has never been identified in extracts of infected cells or in any tail assembly intermediate. In order to overcome this difficulty, we expressed gp3 in a high-efficiency plasmid expression vector and subsequently purified it for further analysis. The N-terminal sequence of the purified protein showed that the initial methionine had been removed. Variant C-terminal amino acid sequences were resolved by determining the cysteine content of the protein. The molecular mass of 20.6 kDa for the pure protein was confirmed by Western blotting, using a specific anti-gp3 serum for which the purified protein was the immunogen. We also demonstrated, for the first time, the physical presence of gp3 in the mature T4 phage particle and localized it to the tail tube. By finding a nonleaky, nonpermissive host for a gene 3 mutant, we could clearly demonstrate a new phenotype: the slow, aberrant elongation of the tail tube in the absence of gp3.

Bacteriophage-like particles associated with the gene transfer agent of methanococcus voltae PS.

The methanogenic archaeobacterium Methanococcus voltae (strain PS) is known to produce a filterable, DNase-resistant agent (called VTA, for voltae transfer agent), which carries very small fragments (4400 bp) of bacterial DNA and is able to transduce bacterial genes between derivatives of the strain. Examination by electron microscopy of two preparations of VTA that were concentrated and partially purified by different methods showed virus-like particles with isometric heads, about 40 nm in diameter, and with 61 nm long tails. These particles co-sedimented with the minute bacteriophage φX174 in a sucrose density gradient.

Tail length determination in bacteriophage T4.

This report identifies a protein that regulates tail length in bacteriophage T4. Earlier work (Duda et al., 1990) suggested that the gene 29 protein could be involved in T4 tail length determination as a "template" or "tape-measure", similar to that proposed for the gene H protein in bacteriophage lambda. We have altered the length of a recombinant gene 29 by constructing deletions and duplications in different parts of the gene. Each of these constructs was incorporated into the high-level expression vector, pET-11d. Seven plasmids with different lengths of gene 29 were made and used in complementation studies. We have found that the length of the tail can be decreased by deleting the C-terminal part of gene 29 or increased by forming duplications in different parts of this gene, and that the length of the tail can be proportional to the size of the engineered protein. Unlike phage lambda, plasmids with deletions in the middle of gene 29 or from the N-terminal end produced correspondingly smaller but inactive gene 29 protein and no viable phage were formed. Our results show that alterations in the length of gene 29 protein proportionately alters tail length, and argue strongly for a scheme in which 29 protein is a ruler or template that determines tail length during tail assembly.

Expression of plasmid-encoded structural proteins permits engineering of bacteriophage T4 assembly.

A complementation system for studying bacteriophage T4 tail assembly has been developed and used to test the effects of nonviable mutations on the function of a specific T4 tail protein, gp48. The complementation system assays the assembly function of gp48 without requiring that viable phage be produced, circumventing the operational problems of maintaining nonviable mutants of this lytic bacteriophage. The protein to be tested was preexpressed from cloned genes in a host cell prior to infection with the challenge phage. Assembly activity was assayed by monitoring the conversion of one tail assembly intermediate, the baseplate lacking gp48, into baseplates containing gp48 or into tube baseplates (or sheathed tails) assembled from such baseplates. Specific incorporation of gp48 into these structures was confirmed using gp48-specific antiserum, and the same serum was used in direct immunoelectron microscopy experiments to localize gp48 to the baseplate-proximal end of the T4 tail tube, at the site where the tube and sheath bind to the baseplate. The protein gp48 has been previously shown to be a baseplate protein, as well as a tail-tube-associated protein, and was tested for a possible role as a tail-length tape-measure protein. Tests with a deleted variant of gp48 were inconclusive because the protein was inactive. A variant of gp48, 20% longer than wild-type protein due to an internal duplication, was found to be partly functional in our assembly complementation system. This abnormally elongated protein allows several assembly steps to proceed, including the assembly of normal length T4 tails, implying that it does not specify tail length. The insertion-duplication variant of gp48 appears to have a defect in its interaction with the tail sheath protein, leading to abnormal sheath contraction.

Potential length determiner and DNA injection protein is extruded from bacteriophage T4 tail tubes in vitro.

Bacteriophage T4 tails contain a set of extended protein molecules in the central channel of the tail tube through which the DNA must exit during infection. Treatment of tails with guanidine hydrochloride separates the baseplates, leaving the tail tube and several specific tube-associated proteins. Methods were developed to purify these structures. Using specific antisera, immunoblotting, and electrophoretic analysis, these structures were shown to contain proteins gp19, 29, and 48. Electron microscopy showed specifically defined stain penetration into the tail tube, a bulge at one end, and a short fiber extruded from the tube. These structures could be removed by proteases but the gp19 tube itself was resistant. Structural studies of tails and intact phage show that the bulge and fiber are at the end of the tube that interacts with the cell membrane during infection. Since the fiber did not protrude from baseplates or from incomplete (short) tube-baseplates, we propose that it is first assembled as a compact structure formed of six copies of a tube-associated protein, which elongates during tail tube formation to fill the central channel, span the length of the tube, and regulate its length. We suggest that the exit of this fiber during infection signals DNA ejection.