Search PubMed⌕ Search

Biomedical subjects

S C Harrison

Publications and source records attributed to S C Harrison.

At least 145 records · Page 8Linked to original sources
PubMed1986

Structure and assembly of turnip crinkle virus. II. Mechanism of reassembly in vitro.

Dissociation of turnip crinkle virus (TCV) at elevated pH and ionic strength produces free dimers of the coat protein and a ribonucleoprotein complex that contains the viral RNA, six coat-protein subunits, and the minor protein species, p80 (a covalently linked coat-protein dimer). This "rp-complex" is stable for several days in high salt at pH 8.5. Reassembly of TCV can be accomplished under physiological conditions, using isolated coat protein and either rp-complex or protein-free RNA. If rp-complex is used in reassembly, the same subunits remain bound to RNA on subsequent dissociation; if free RNA is used, rp-complex is regenerated. In both cases, the assembly is selective for viral RNA in competition experiments with heterologous RNA. Electron microscopy shows that assembly proceeds by continuous growth of a shell from an initiating structure, rather than by formation of distinct intermediates. We suggest that rp-complex is the initiating structure, suggest a model based on the organization of the TCV particle, and propose a mechanism for TCV assembly.

Capsid↗
PubMed1986

Structure of turnip crinkle virus. III. Identification of a unique coat protein dimer.

The minor structural protein (p80), found in about one copy per virion in turnip crinkle virus (TCV), is shown by amino acid analysis and peptide mapping to be a covalent dimer of the major coat protein (p40). The covalent linkage occurs near the N termini of the crosslinked chains. These data suggest that TGV and related viruses contain 178 copies of p40 (89 non-covalent dimers) and one copy of p80 (covalent dimer of two additional p40 chains). The presence of p80 in the salt-stable RNA-protein complex formed when TCV dissociates, as described in an accompanying paper, indicates that the covalent modification affects binding to RNA. We suggest that p80 might be the final dimer to be incorporated into the shell and that it might also be the site for initiation of uncoating.

Amino Acids↗
PubMed1986

Protein, DNA, and virus crystallography with a focused imaging proportional counter.

A set of programs has been developed for rapid collection of x-ray intensity data from protein and virus crystals with a commercially available two-dimensional focused geometry electronic detector. The detector is compact and portable, with unusually high spatial resolution comparable to that used in oscillation photography. It has allowed x-ray data collection on weakly diffracting crystals with large unit cells, as well as more conventional "diffractometer-quality" crystals. The quality of the data is compared with that from oscillation photography and automated diffractometry in the range of unit cells from 96.3 to 383.2 angstroms. Isomorphous and anomalous difference Pattersons, based on detector data, are shown for a variable surface glycoprotein mercury derivative and for a repressor-DNA bromine derivative, which has been solved at 7 angstroms with detector data only.

Computers↗
PubMed1986

Configuration of clathrin trimers: evidence from electron microscopy.

We used a combination of electron microscopic techniques--negative staining, glycerol spraying with rotary shadowing, and quick-freezing followed by deep-etching--to study the configuration of clathrin trimers. All three approaches provide images indicating that the molecule is nonplanar and rather puckered at its center. Viewed from the convex (cytoplasmic) side, its arms display a consistent clockwise slew at their proximal/distal hinge. The most flexible part of the heavy chain may be the links between the distal portion of the leg and the terminal domain.

Clathrin↗
PubMed1985

DNA gyrase and its complexes with DNA: direct observation by electron microscopy.

Electron microscopy of DNA gyrase holoenzyme, of gyrase A subunits, and of the complexes of both species with DNA enables us to deduce the relative locations of subunits in the holoenzyme and to indicate a plausible path for DNA complexed with gyrase. The structural results are discussed in terms of certain models for directional DNA strand transport.

Binding Sites↗
PubMed1985

Is there a single pathway for the folding of a polypeptide chain?

We argue that folding of the compact domains of proteins can occur with adequate rapidity in the absence of a unique directed mechanism, provided that native-like local structure dominates the folding process. We further suggest that the evolution of amino acid sequences should favor multiple paths to the folded state. Existing physicochemical and mutational data are not inconsistent with a many-pathway model. The analogy of a jigsaw puzzle, with multiple routes to a unique solution, appears to be particularly apt.

Amino Acid Sequence↗
PubMed1984

Structure of tomato bushy stunt virus. V. Coat protein sequence determination and its structural implications.

We report the chemically determined sequence of most of the polypeptide chain of the coat protein of tomato bushy stunt virus. Peptide locations have been determined by comparison with the high-resolution electron density map from X-ray crystallographic analysis as well as by conventional chemical overlaps. Three small gaps remain in the 387-residue sequence. Positively charged side-chains are concentrated in the N-terminal part of the polypeptide (the R domain) as well as on inward-facing surfaces of the S domain. There is homology of S-domain sequences with structurally corresponding residues in southern bean mosaic virus.

Amino Acid Sequence↗
PubMed1984

Cocrystals of the DNA-binding domain of phage 434 repressor and a synthetic phage 434 operator.

The amino-terminal domain of the phage 434 repressor forms cocrystals with a synthetic phage 434 operator. The cocrystals diffract to at least 4 A, and x-ray crystallographic analysis of them is in progress. An analysis of the packing in the cocrystals shows that complexes consisting of dimers of amino-terminal domain bound specifically to operators are stacked end to end in longer protein-DNA rods parallel to the unit cell body diagonals. The DNA in the complexes has 10.5 base pairs per turn and a rise per base of 3.26 A--values consistent with B-form DNA--indicating that DNA is neither unwound nor overwound by bound repressor. The packing analysis suggests an approach that might facilitate the cocrystallization of other DNA-binding proteins with the DNA they recognize.

Base Composition↗
PubMed1984

Structural domains of clathrin heavy chains.

We used a combination of electron microscopy and proteolytic dissection to study the substructure of the clathrin trimer. The fragments of a heavy chain generated by limited proteolysis of cages were examined by rotary shadowing after disassembly. Correlation of lengths and molecular weights allowed us to map certain cleavage points along an arm and to assign them to positions in a model for a cage. We found that a particularly stable fragment of 52,000-59,000 Mr (depending on the enzyme) corresponded to the knob-like terminal domain at the tip of each arm.

Animals↗
PubMed1983

Packaging of DNA into bacteriophage heads: a model.

A model is suggested for the geometry of DNA entry into a bacteriophage head. It accounts for recent observations indicating absence of a unique, ordered sequence of windings in the packaged DNA.

Bacteriophages↗
PubMed1983

Structure of tomato busy stunt virus IV. The virus particle at 2.9 A resolution.

The structure of tomato bushy stunt virus has been determined crystallographically to 2.9 A resolution. Details are presented of both the molecular structure and the methods by which it has been solved. The icosahedrally symmetric viral shell is composed of 180 protein subunits (Mr 43,000), with three similar but distinct modes of subunit bonding. This capacity for alternative packing is due to localized flexibility in the folded polypeptide (hinges between domains) and to multiple conformations for surface side-chains. The polypeptide backbone has an essentially invariant fold within a compact domain. A mechanism for correct positioning of the different modes of subunit interaction is evident from the structure of the TBSV particle. Thirty-five residues of the polypeptide chain fold in an ordered way on 60 of the 180 subunits, forming an internal framework. Interaction of folded domains with this framework permits accuracy of long-range geometry (correct curvature and closure) to be determined by unambiguous switching between alternative local contact angles. RNA packs tightly into the particle interior. Protein-RNA interactions occur through parts of the subunit that are flexibly linked to the well-ordered domains of the shell. This variable interaction imposes minimum restrictions on the folding of the RNA chain.

Amino Acid Sequence↗
PubMed1983

Divalent cation sites in tomato bushy stunt virus. Difference maps at 2-9 A resolution.

Difference electron density maps, using as few as four 1/2 degrees oscillation photographs, have been computed for tomato bushy stunt virus crystals soaked in EDTA. GdCl3 and silicotungstate. The maps define a double divalent cation site, responsible for regulating expansion of the virus particle, as well as sites for binding tungstate anions.

Binding Sites↗
PubMed1983

Location and distribution of the light chains in clathrin trimers.

Mouse monoclonal antibodies have been made that are specific for one of the light chains (LC-A) of calf brain clathrin. The determinant they recognize has been mapped by immunoelectron microscopy and shown to lie near the center of a clathrin trimer. Quantitative immunoprecipitation experiments with one of the monoclonal antibodies suggests that, despite the overall 1:2 stoichiometry, the light chain composition of an individual clathrin trimer is nearly random.

Animals↗
PubMed1981

Protein organization in clathrin trimers.

We have prepared a homogeneous, soluble 8.6S species ("8.6S clathrin") from calf-brain coated vesicles. Crosslinking experiments show that this 8.6S clathrin is composed of three heavy chains (molecular weight 180,000) and three light chains (molecular weights 33,000 and 36,000). Each heavy chain is in close contact with a single light chain, and the light chains appear not to be in contact with each other. Intact 8.6S clathrin can reassemble into cages without participation of additional protein species.

Animals↗