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Publications and source records attributed to S C Harrison.
The crystal structure of phage 434 Cro protein has been determined and refined against 2.35 A data to an R-factor of 19.5%. The protein comprises five alpha-helices and shows the helix-turn-helix motif found in other repressor proteins.
The crystal structure of the amino-terminal domain of phage 434 repressor has been solved using molecular replacement methods and refined to an R-factor of 19.3% against data to 2.0 A resolution. The protein comprises five short alpha-helices. Two of these form a helix-turn-helix motif, very similar to those found in related proteins. The protein is remarkably similar to the Cro protein from the same phage.
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Coated vesicles were purified from human placenta by sedimentation, isopycnic centrifugation, and gel filtration. Quantitative Western blotting of the endogenous transferrin receptor (tfR) demonstrated the presence, on average, of roughly one receptor per vesicle. TfR appeared undersaturated with transferrin. After solubilizing vesicles in nonionic detergent, we looked for evidence of tfR interactions with other proteins. Solubilized tfR had an unexpectedly high mobility by gel filtration, apparently resulting from its self-association. This property was not seen in purified tfR or in tfR from a different cell fraction. The tfR complexes, though noncovalent, were largely resistant to conditions that disassemble coat proteins, and they did not appear to contain any other protein species.
The repressors of temperate bacteriophages such as 434 and lambda control transcription by binding to a set of DNA operator sites. The different affinity of repressor for each of these sites ensures efficient regulation. High-resolution x-ray crystallography was used to study the DNA-binding domain of phage 434 repressor in complex with a synthetic DNA operator. The structure shows recognition of the operator by direct interactions with base pairs in the major groove, combined with the sequence-dependent ability of DNA to adopt the required conformation on binding repressor. In particular, a network of three-centered bifurcated hydrogen bonds among base pairs in the operator helps explain why 434 repressor prefers certain sites over others. These bonds, which stabilize the conformation of the bound DNA, can form only with certain sequences.
Human transferrin receptor (tfR) is a covalent homodimer of 90-kDa transmembrane subunits, which transits an endocytotic pathway involving exposure to low pH. Digestion of purified tfR at neutral pH generates a soluble noncovalent dimer of 70-kDa fragment subunits containing 95% of the extracellular tfR sequence, including the transferrin binding sites. Below pH 6, the 70-kDa fragment undergoes a conformational transition, which causes reversible association of the dimers in solution. Transferrin binding prevents both the conformational transition and the self-association. We suggest that tfR clustering in acidic compartments results from self-association due to a conformational change that is sensitive to transferrin binding. This and other observations support a concentration mechanism based on interactions between ectodomains in intracellular lumina.
Comparison of the crystal structure of a complex of the phage 434 Cro protein and a synthetic DNA operator with the complex of the same operator and the 434 repressor DNA-binding domain shows different DNA conformations in the two structures. Binding of the protein determines the precise conformation of the DNA in each case.
Human transferrin receptor is a disulfide-linked homodimer of 90-kDa glycoprotein subunits, capable of binding two transferrins. We report a new high yield affinity purification protocol for transferrin receptor from placenta which produces 3-4 mg of highly purified protein. Trypsin cleaves the protein at arginine-121, producing a stable fragment that contains 95% of the extracytoplasmic sequence; similar fragments are produced by several other proteases. The tryptic fragment is a nondisulfide-linked dimer in solution and binds two transferrin molecules. The dimensions of both the dimer fragment and its complex with transferrin are estimated by gel filtration.
The structure of a complex between the DNA-binding domain of phage 434 repressor and a 14 base-pair synthetic DNA operator reveals the molecular interactions important for sequence-specific recognition. A set of contacts with DNA backbone, notably involving hydrogen bonds between peptide-NH groups and DNA phosphates, position the repressor and fix the DNA configuration. Direct interactions between amino acid side chains and DNA bases involve nonpolar van der Waals contacts as well as hydrogen bonds. The structures of the repressor domain and of the 434 cro protein are extremely similar. There appear to be no major conformational changes in the proteins when they bind to DNA.
The affinity of the Escherichia coli phage 434 operator for phage 434 repressor is affected by changes in the sequence of the noncontacted base pairs near the operator's center. The results presented here show that base composition near the center of the operator affects the operator's affinity for repressor by altering the ease with which the operator can be overtwisted into the proper configuration for complex formation. We show that both DNA flexibility and repressor flexibility influence the strength of the repressor-operator interaction: an operator with a single-strand nick at its center has a higher affinity for repressor than does the intact operator: and a repressor bearing a mutation that results in a relaxed dimer interaction is less sensitive than is wild type to changes in the flexibility of the operator. We show that the effect of noncontacted base pairs on operator affinity is independent of the slight overall bend of the operator seen in the repressor-operator complex. Central sequence effects on affinity for repressor are independent of the identity of adjacent base pairs, suggesting that the structure of the individual base pairs, not interactions between them, are responsible for the different torsional rigidities of different operators.
A technique is described for performing in vitro muscle strip studies using samples of detrusor muscle taken by means of endoscopic biopsy forceps. This study of pig bladder muscle showed that the nerve mediated and drug induced responses of the muscle showed that the nerve mediated and drug induced responses of the muscle biopsies were similar to those produced by traditionally dissected muscle strips. The importance of continued access to human material for pharmacological studies is discussed.
Crystals have been obtained of the bacteriophage 434 Cro protein bound to a synthetic DNA operator. An analysis of the packing shows that the complexes stack end-to-end along crystallographic axis, forming long rods with non-crystallographic 11(3) screw symmetry. The average number of DNA base-pairs per turn is 10.27, which is somewhat more overwound as compared with the 434 repressor-DNA crystals of Anderson et al. Diffraction extends to 3 A along the rod direction and to 5 A in perpendicular directions.
The clathrin light chains fall into two major classes, LCA and LCB. In an intact clathrin triskelion, one light chain, of either class, is bound to the proximal segment of a heavy chain leg. Analysis of rat brain and liver complementary DNA clones for LCA and LCB shows that the two light chain classes are closely related. There appear to be several members of each class having deletions of varying length aligned at the same position. A set of ten heptad elements, characteristic of alpha-helical coiled coils, is a striking feature of the central part of each derived amino acid sequence. These observations suggest a model in which the alpha-helical segment mediates binding to clathrin heavy chains and the amino- and carboxyl-terminal segments mediate interactions with other proteins. They also suggest an explanation for the observed tissue-dependent size variation for members of each class.
The structure of the turnip crinkle virus (TCV) coat protein and coat protein gene has been examined by cDNA cloning, nucleotide sequencing and high-resolution mRNA mapping. We have cloned a 1450-nucleotide cDNA fragment, representing the 3' end of the TCV genome, using genomic RNA polyadenylated in vitro as the reverse transcriptional template. Nucleic acid sequence analysis reveals the presence of a 1053 nucleotide open reading frame capable of encoding a protein of 38,131 Mr, identified as the coat protein subunit. The 1446 base subgenomic mRNA for the coat protein, mapped using high-resolution primer extension techniques, contains a 137 nucleotide leader sequence upstream from the initiation codon. We have characterized a second subgenomic RNA of approximately 1700 bases, roughly 250 nucleotides longer than the 1446 base species in the 5' direction. No TCV-related RNAs are polyadenylated in vivo. The derived amino acid sequence of the TCV coat protein has been built into the 3.2 A resolution electron density map of TCV reported in paper I of this series. We describe here some of the important features of the structure. Alignment of the three-dimensional structures of tomato bushy stunt virus and southern bean mosaic virus shows significant sequence relationships in the arms and S domains, although the conserved residues do not appear to have any special role in stabilizing the beta-barrel fold or in mediating subunit interactions. The sequences of TCV and carnation mottle virus can be aligned. Comparisons among the four are discussed in terms of the organization of the S domain.
We have deduced the 1675-amino acid sequence of rat clathrin heavy chain from cDNA clones and predict a protein of Mr 191,569. We have established the polarity of the heavy chain and assigned sequence positions to several structural landmarks of the clathrin leg. The terminal domain at the distal end of the clathrin leg is at the amino terminus of the heavy chain. It is connected to the distal segment by a flexible "link" from Tyr-479 to Arg-523. There is an unusual sequence at the carboxyl terminus that may form the globular projection at the vertex of the clathrin trimer. We suggest that a possible site of heavy-chain-light-chain interaction is located in the proximal segment. Comparison with other partially sequenced mammalian clathrin heavy chains shows that the primary structure is highly conserved. The heavy chain is unrelated to other classes of structural proteins.
Nineteen cases of idiopathic urethritis affecting male children are presented. Details of the symptoms and the course of the illness are given along with recommendations regarding management. Symptoms usually occurred in a recurrent pattern and lasted, on average, 2 years. The diagnosis was made on clinical grounds after bacterial infection had been excluded; urethroscopy was used to confirm the diagnosis when necessary. The prognosis was generally good, although no specific treatment is available.
In vitro pharmacological studies were performed on endoscopic detrusor biopsies from patients with bladder outflow obstruction. Urodynamic studies had been undertaken to detect the presence of bladder instability. Muscle strips from patients with instability demonstrated supersensitivity to acetylcholine and reduction in nerve mediated responses, as compared with strips from stable bladders. These changes are interpreted as suggesting the presence of cholinergic denervation in obstructed patients with bladder instability.
The structure of turnip crinkle virus has been determined at 3.2 A resolution, using the electron density of tomato bushy stunt virus as a starting point for phase refinement by non-crystallographic symmetry. The structures are very closely related, especially in the subunit arm and S domain, where only small insertions and deletions and small co-ordinate shifts relate one chain to another. The P domains, although quite similar in fold, are oriented somewhat differently with respect to the S domains. Understanding of the structure of turnip crinkle virus has been important for analyzing its assembly, as described in an accompanying paper.