Martha N. Simon

Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels.

The DNA of bacteriophage T7 is cut into seven unique fragments by the restriction endonuclease Dpn II (or the equivalent Mbo I), 19 fragments by Hpa I, and eight additional fragments by the combination of the two enzymes. The relative location of each fragment in the T7 DNA has been determined by a combination of techniques. If it is assumed that the length of any DNA molecule equals the sum of the lengths of the fragments produced from it by cleavage, and that electrophoretic mobility through agarose gels is a smooth function of the length of the DNA, then the known relationships between fragments provide enough conditions to define accurately the relative molecular weight of each fragment in the set. Absolute molecular weights are based on that of full-length T7 DNA. The fragments provide a convenient set of length standards covering the entire range from about 100 to 40,000 base-pairs (the length of T7 DNA). A horizontal slab gel system for electrophoresis on agarose gels is described. In this system, gels of very low concentrations do not distort during electrophoresis and accurate relative mobilities of large DNAs are obtained. Excellent resolution can be obtained for DNAs of molecular weights up to at least 26·5×10 6 , a difference of less than 10% being readily resolved even for molecules of this size. Agarose and polyacrylamide gels can be prepared in alkaline solvents that denature native DNA and completely unfold the single strands. The fragments of T7 DNA have the same relative mobilities whether subjected to electrophoresis as single strands in alkaline gels or as double-stranded DNA in neutral gels, and resolution is comparable in the two states. Thus, electrophoresis in alkaline gels can provide accurate molecular weights for linear, single-stranded DNAs, and should be useful in analyzing DNA for single-strand breaks, depurinations or topological differences such as ring forms. In both neutral and alkaline gels, the relative mobilities of DNAs shorter than about 1000 base-pairs (or bases) are essentially insensitive to changes in voltage gradient, at least over the range of voltage gradients commonly employed. However, relative mobilities become increasingly sensitive to voltage gradient the larger the DNA, with DNAs longer than about 20,000 base-pairs (or bases) being severely affected. This effect is probably due to the ease with which large DNA molecules can be deformed from their equilibrium conformations, thus permitting them to penetrate channels in the gel that would exclude them in their unperturbed conformations. As a practical matter, this means that low voltage gradients must be used for separations of large DNAs by gel electrophoresis.

α-Synuclein, especially the Parkinson's disease-associated mutants, forms pore-like annular and tubular protofibrils

Two mutations in the alpha-synuclein gene (A30P and A53T) have been linked to autosomal dominant early-onset Parkinsons disease (PD). Both mutations promote the formation of transient protofibrils (prefibrillar oligomers), suggesting that protofibrils are linked to cytotoxicity. In this work, the effect of these mutations on the structure of alpha-synuclein oligomers was investigated using electron microscopy and digital image processing. The PD-linked mutations (A30P and A53T) were observed to affect both the morphology and the size distribution of alpha-synuclein protofibrils (measured by analytical ultracentrifugation and scanning transmission electron microscopy). The A30P variant was observed to promote the formation of annular, pore-like protofibrils, whereas A53T promotes formation of annular and tubular protofibrillar structures. Wild-type alpha-synuclein also formed annular protofibrils, but only after extended incubation. The formation of pore-like oligomeric structures may explain the membrane permeabilization activity of alpha-synuclein protofibrils. These structures may contribute to the pathogenesis of PD.

The stoichiometry of Gag protein in HIV-1

The major structural components of HIV-1 are encoded as a single polyprotein, Gag, which is sufficient for virus particle assembly. Initially, Gag forms an approximately spherical shell underlying the membrane of the immature particle. After proteolytic maturation of Gag, the capsid (CA) domain of Gag reforms into a conical shell enclosing the RNA genome. This mature shell contains 1,000–1,500 CA proteins assembled into a hexameric lattice with a spacing of 10 nm. By contrast, little is known about the structure of the immature virus. We used cryo-EM and scanning transmission EM to determine that an average (145 nm diameter) complete immature HIV particle contains ∼5,000 structural (Gag) proteins, more than twice the number from previous estimates. In the immature virus, Gag forms a hexameric lattice with a spacing of 8.0 nm. Thus, less than half of the CA proteins form the mature core.

Mixtures of Wild-type and a Pathogenic (E22G) Form of Aβ40 in Vitro Accumulate Protofibrils, Including Amyloid Pores ☆

Although APP mutations associated with inherited forms of Alzheimers disease (AD) are relatively rare, detailed studies of these mutations may prove critical for gaining important insights into the mechanism(s) and etiology of AD. Here, we present a detailed biophysical characterization of the structural properties of protofibrils formed by the Arctic variant (E22G) of amyloid-beta protein (Abeta40(ARC)) as well as the effect of Abeta40(WT) on the distribution of the protofibrillar species formed by Abeta40(ARC) by characterizing biologically relevant mixtures of both proteins that may mimic the situation in the heterozygous patients. These studies revealed that the Arctic mutation accelerates both Abeta oligomerization and fibrillogenesis in vitro. In addition, Abeta40(ARC) was observed to affect both the morphology and the size distribution of Abeta protofibrils. Electron microscopy examination of the protofibrils formed by Abeta40(ARC) revealed several morphologies, including: (1) relatively compact spherical particles roughly 4-5 nm in diameter; (2) annular pore-like protofibrils; (3) large spherical particles 18-25 nm in diameter; and (4) short filaments with chain-like morphology. Conversion of Abeta40(ARC) protofibrils to fibrils occurred more rapidly than protofibrils formed in mixed solutions of Abeta40(WT)/Abeta40(ARC), suggesting that co-incubation of Abeta40(ARC) with Abeta40(WT) leads to kinetic stabilization of Abeta40(ARC) protofibrils. An increase in the ratio of Abeta(WT)/Abeta(MUT(Arctic)), therefore, may result in the accumulation of potential neurotoxic protofibrils and acceleration of disease progression in familial Alzheimers disease mutation carriers.

Three-Dimensional Analysis of Budding Sites and Released Virus Suggests a Revised Model for HIV-1 Morphogenesis

Current models of HIV-1 morphogenesis hold that newly synthesized viral Gag polyproteins traffic to and assemble at the cell membrane into spherical protein shells. The resulting late-budding structure is thought to be released by the cellular ESCRT machinery severing the membrane tether connecting it to the producer cell. Using electron tomography and scanning transmission electron microscopy, we find that virions have a morphology and composition distinct from late-budding sites. Gag is arranged as a continuous but incomplete sphere in the released virion. In contrast, late-budding sites lacking functional ESCRT exhibited a nearly closed Gag sphere. The results lead us to propose that budding is initiated by Gag assembly, but is completed in an ESCRT-dependent manner before the Gag sphere is complete. This suggests that ESCRT functions early in HIV-1 release--akin to its role in vesicle formation--and is not restricted to severing the thin membrane tether.

Beta-helix model for the filamentous haemagglutinin adhesin of Bordetella pertussis and related bacterial secretory proteins

Bordetella pertussis establishes infection by attaching to epithelial cells of the respiratory tract. One of its adhesins is filamentous haemagglutinin (FHA), a 500‐Å‐long secreted protein that is rich in β‐structure and contains two regions, R1 and R2, of tandem 19‐residue repeats. Two models have been proposed in which the central shaft is (i) a hairpin made up of a pairing of two long antiparallel β‐sheets; or (ii) a β‐helix in which the polypeptide chain is coiled to form three long parallel β‐sheets. We have analysed a truncated variant of FHA by electron microscopy (negative staining, shadowing and scanning transmission electron microscopy of unstained specimens): these observations support the latter model. Further support comes from detailed sequence analysis and molecular modelling studies. We applied a profile search method to the sequences adjacent to and between R1 and R2 and found additional ‘covert’ copies of the same motifs that may be recognized in overt form in the R1 and R2 sequence repeats. Their total number is sufficient to support the tenet of the β‐helix model that the shaft domain – a 350 Å rod – should consist of a continuous run of these motifs, apart from loop inserts. The N‐terminus, which does not contain such repeats, was found to be weakly homologous to cyclodextrin transferase, a protein of known immunoglobulin‐like structure. Drawing on crystal structures of known β‐helical proteins, we developed structural models of the coil motifs putatively formed by the R1 and R2 repeats. Finally, we applied the same profile search method to the sequence database and found several other proteins – all large secreted proteins of bacterial provenance – that have similar repeats and probably also similar structures.

Physical mapping of the early region of bacteriophage T7 DNA.

Abstract Heteroduplex mapping of bacteriophage T7 deletions, together with genetic and electrophoretie analysis of T7 RNAs and proteins, has produced a detailed physical map of a substantial segment of T7 DNA, including most of the early region. Position in the T7 molecule is given as percentage of the length of wild type T7 DNA, measured from the left end. The signal that stops transcription by Escherichia coli RNA polymerase at the end of the early region is located near position 20.2. The five early messenger RNAs seem to occupy fully the region between 1.8 and 20.2, with little if any gap between molecules, and the ends of these RNAs have been mapped at positions 1.8, 3.4, 8.0, 15.5, 17.1 and 20.2. When the stop signal is deleted, transcription by host RNA polymerase continues on and the next RNA ends at position 30.1. The positions of some early T7 proteins, as well as one protein to the right of the stop signal, have also been determined. The mapping of the 0.3 and 0.7 proteins is not straightforward, and may indicate that these proteins are somehow processed during or after synthesis. The DNA between positions 2.8 and 8.1, and between positions 15.2 and 23.5, is not essential for growth of the phage, since it can be eliminated by deletions. Genes 0.7 , 1.1 and 1.3 can be deleted entirely, as can the usual origin of replication of T7 DNA, determined by Dressier et al . (1972) to be near position 17. The molecular weights of the early T7 RNAs range between 208,000 and 975,000 and give a convenient spread to use as standards for electrophoresis of RNAs in polyacrylamide gels.

Characterization of Rous sarcoma virus Gag particles assembled in vitro.

ABSTRACT Purified retrovirus Gag proteins or Gag protein fragments are able to assemble into virus-like particles (VLPs) in vitro in the presence of RNA. We have examined the role of nucleic acid and of the NC domain in assembly of VLPs from a Rous sarcoma virus (RSV) Gag protein and have characterized these VLPs using transmission electron microscopy (TEM), scanning TEM (STEM), and cryoelectron microscopy (cryo-EM). RNAs of diverse sizes, single-stranded DNA oligonucleotides as small as 22 nucleotides, double-stranded DNA, and heparin all promoted efficient assembly. The percentages of nucleic acid by mass, in the VLPs varied from 5 to 8%. The mean mass of VLPs, as determined by STEM, was 6.5 × 107 Da for both RNA-containing and DNA oligonucleotide-containing particles, corresponding to a stoichiometry of about 1,200 protein molecules per VLP, slightly lower than the 1,500 Gag molecules estimated previously for infectious RSV. By cryo-EM, the VLPs showed the characteristic morphology of immature retroviruses, with discernible regions of high density corresponding to the two domains of the CA protein. In spherically averaged density distributions, the mean radial distance to the density corresponding to the C-terminal domain of CA was 33 nm, considerably smaller than that of equivalent human immunodeficiency virus type 1 particles. Deletions of the distal portion of NC, including the second Zn-binding motif, had little effect on assembly, but deletions including the charged residues between the two Zn-binding motifs abrogated assembly. Mutation of the cysteine and histidine residues in the first Zn-binding motif to alanine did not affect assembly, but mutation of the basic residues between the two Zn-binding motifs, or of the basic residues in the N-terminal portion of NC, abrogated assembly. Together, these findings establish VLPs as a good model for immature virions and establish a foundation for dissection of the interactions that lead to assembly.

Structure of Hepatitis E Virion-sized Particle Reveals an RNA-dependent Viral Assembly Pathway

Hepatitis E virus (HEV) induces acute hepatitis in humans with a high fatality rate in pregnant women. There is a need for anti-HEV research to understand the assembly process of HEV native capsid. Here, we produced a large virion-sized and a small T=1 capsid by expressing the HEV capsid protein in insect cells with and without the N-terminal 111 residues, respectively, for comparative structural analysis. The virion-sized capsid demonstrates a T=3 icosahedral lattice and contains RNA fragment in contrast to the RNA-free T=1 capsid. However, both capsids shared common decameric organization. The in vitro assembly further demonstrated that HEV capsid protein had the intrinsic ability to form decameric intermediate. Our data suggest that RNA binding is the extrinsic factor essential for the assembly of HEV native capsids.

Structure of the Filamentous Phage pIV Multimer by Cryo-electron Microscopy

The homo-multimeric pIV protein constitutes a channel required for the assembly and export of filamentous phage across the outer membrane of Escherichia coli. We present a 22 A-resolution three-dimensional reconstruction of detergent-solubilized pIV by cryo-electron microscopy associated with image analysis. The structure reveals a barrel-like complex, 13.5 nm in diameter and 24 nm in length, with D14 point-group symmetry, consisting of a dimer of unit multimers. Side views of each unit multimer exhibit three cylindrical domains named the N-ring, the M-ring and the C-ring. Gold labeling of pIV engineered to contain a single cysteine residue near the N or C terminus unambiguously identified the N-terminal region as the N-ring, and the C-terminal region was inferred to make up the C-ring. A large pore, ranging in inner diameter from 6.0 nm to 8.8 nm, runs through the middle of the multimer, but a central domain, the pore gate, blocks it. Moreover, the pore diameter at the N-ring is smaller than the phage particle. We therefore propose that the pIV multimer undergoes a large conformational change during phage transport, with reorganization of the central domain to open the pore, and widening at the N-ring in order to accommodate the 6.5 nm diameter phage particle.