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ABSTRACT: Oligomerization by the formation of alpha-helical bundles is common in many proteins. The crystal structure of a parallel pentameric coiled coil, constituting the oligomerization domain in the cartilage oligomeric matrix protein (COMP), was determined at 2.05 angstroms resolution. The same structure probably occurs in two other extracellular matrix proteins, thrombospondins 3 and 4. Complementary hydrophobic interactions and conserved disulfide bridges between the alpha helices result in a thermostable structure with unusual properties. The long hydrophobic axial pore is filled with water molecules but can also accommodate small apolar groups. An "ion trap" is formed inside the pore by a ring of conserved glutamines, which binds chloride and probably other monatomic anions. The oligomerization domain of COMP has marked similarities with proposed models of the pentameric transmembrane ion channels in phospholamban and the acetylcholine receptor.
Science 12/1996; 274(5288):761-5. · 31.20 Impact Factor
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ABSTRACT: Cartilage oligomeric matrix protein (COMP) is a pentameric glycoprotein of the thrombospondin family found in cartilage and tendon. Self-association of COMP is achieved through the formation of a five-stranded alpha-helical bundle that involves 64 N-terminal residues (from 20 to 83). The complex is further stabilized by the interchain disulfide bonds between cysteines 68 and 71. We have prepared, by expression in Escherichia coli, several peptides of different lengths from the N-terminal region of COMP and studied their amenability to crystallization. Crystals of the best quality were obtained with a peptide spanning COMP residues 28-72. This peptide forms disulfide linked pentamers with 87% of alpha-helical structure. Crystals were grown by the hanging drop vapor diffusion method, using polyethylene glycol 1500 as a precipitant. The crystals belong to space group P2(1) with unit cell dimensions a = 38.47 angstroms, b = 49.47 angstroms, c = 54.98 angstroms, beta = 103.84 degrees and contain one pentamer per asymmetric unit. They diffract strongly to at least 1.8 angstrom resolution.
Proteins Structure Function and Bioinformatics 03/1996; 24(2):259-62. · 3.39 Impact Factor
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ABSTRACT: We describe a method for construction of hymeric bacteriophage T4 particles displaying foreign polypeptides on their surface. The method is based on our finding that minor T4 fibrous protein fibritin encoded by gene wac (whisker's antigen control) could be lengthened at the C terminus without impairing its folding or binding to the phage particle. The lengthened fibritin gene could easily be transferred into the T4 genome by homologous recombination with a plasmid containing the modified gene wac. The modified gene wac is expressed properly during phage reproduction, and the lengthened fibritin is bound to phage particles. As an example of this type of method, we have obtained the hymeric T4 particles carrying a polypeptide of 53 residues, 45 of which are from the pre-S2 region of hepatitis B virus. The T4 display vector extends currently available display systems.
Virus Genes 02/1995; 10(2):173-7. · 1.85 Impact Factor
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ABSTRACT: The bacteriophage T4 late gene wac (whisker's antigen control) encodes a fibrous protein which forms a collar/whiskers complex. Whiskers function as a helper protein for the long tail fibres assembly and plays a role in regulating retraction of the long tail fibres in response to environmental conditions. In this work we show that expression of the cloned wac gene in Escherichia coli yields a protein oligomer of 53 nm length which we call fibritin, and which is able to complement gpwac T4 particles in vitro. CD spectroscopy of fibritin indicates a 90% alpha-helical content, and scanning calorimetry shows that the protein has several distinct domains. The analysis of the 486 amino acid sequence of fibritin reveals three structural components: a 408 amino acid region that contains 12 putative coiled-coil segments with a canonical heptad (a-b-c-d-e-f-g)n substructure where the "a" and "d" positions are preferentially occupied by apolar residues, and the N and C-terminal domains (47 and 29 amino acid residues, respectively) have no heptad substructure. The distribution of hydrophobic residues within heptads is more similar to a triple than to a double coiled-coil. The alpha-helical segments are separated by short "linker" regions, variable in length, that have a high proportion of glycine and proline residues. Each coiled-coil segment has, on the borders with linker regions, residues that are common to the N and C-terminal caps of the alpha-helices. Full-length and amino-terminally truncated fibritins can be reassembled in vitro after temperature-induced denaturation. Co-assembly of full-length fibritin and the N-terminal deletion mutant, as well as analytical centrifugation, indicates that the protein is a parallel triple-standard alpha-helical coiled-coil. Deletions of various N-terminal portions of fibritin did not block trimerisation but the mutant trimers are unable to bind to T4 particles. The last 18 C-terminal residues of fibritin are required for correct trimerisation of gpwac monomers in vivo. We propose that fibritin might serve as a convenient model for the investigation of folding and assembly mechanisms of alpha-fibrous proteins.
Journal of Molecular Biology 10/1994; 242(4):470-86. · 4.00 Impact Factor
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ABSTRACT: The N-terminal fragment of rat cartilage oligomeric matrix protein (COMP), comprising residues 20-83, was over-expressed in E. coli and purified under non-denaturing conditions. The fragment forms pentamers similar to the assembly domain of the native protein. Its five chains can be covalently linked in vitro by oxidation of cysteines 68 and 71. The fragment adopts a predominantly alpha-helical structure as judged by circular dichroism spectroscopy. On the basis of these findings we propose the model of a five-stranded alpha-helical bundle for the assembly domain of COMP. The studied sequence is conserved in thrombospondins 3 and 4 thus raising the possibility that these proteins are also pentamers.
FEBS Letters 04/1994; 341(1):54-8. · 3.54 Impact Factor
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ABSTRACT: The structural protein, gene product 9 (gp9), of bacteriophage T4 controls baseplate expansion at the first steps of virus attachment onto its host bacterial cell with subsequent tail contraction. Gp9, which has an M(r) of 30.8 kDa and contains 287 amino acids, has been purified from a recombinant Escherichia coli strain and crystallized at 25 degrees C using the hanging drop vapor diffusion method at pH 4.0 with ammonium sulfate as precipitant. The crystals of gp9 belong to the space group R32 with hexagonal cell dimensions a = b = 86.5 A and c = 156.2 A and diffract X-rays to at least 2.7 A. There is one molecule per asymmetric unit.
Journal of Molecular Biology 12/1993; 234(2):493-5. · 4.00 Impact Factor
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Nucleic Acids Research 10/1990; 18(17):5313. · 8.03 Impact Factor
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ABSTRACT: The DNA sequences of genes 9, 10, 11, 12, and wac, which encode the structural proteins in the bacteriophage T4 base plate, were determined. These genes form a single operon which is transcribed in a clockwise direction from a single late promoter in the TATAAATA region located upstream of gene 9 at position -10. A feature of the operon is an overlap between the termination codon of each upstream gene and the initiation codon of its downstream gene. With the exception of gene 10, the open reading frames encode proteins which have a calculated molecular mass close to that obtained experimentally. The reading frame of gene 10 encodes a polypeptide with a calculated molecular mass of 66.2 kDa, which is at least 22 kDa less than that in the phage particle. Thus the mature protein encoded by gene 10 is possibly a product of the fusion of two adjacent phage genes. The hybrid protein may be formed by a frameshift during the translation of messenger RNA at the end of gene 9 or gene 10.
Biomedical science 02/1990; 1(1):55-62.
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Nucleic Acids Research 05/1989; 17(8):3303. · 8.03 Impact Factor
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ABSTRACT: Gene 22 of bacteriophage T4 encodes a major prohead scaffolding core protein of 269 amino acid residues. From its nucleotide sequence the gene product (gp) 22 has a predicted Mr of 29.9 and a pI of 4.3. The protein is rich in charged residues (glutamic acid and lysine) and contains low amounts of proline and glycine and no cysteine residues. We suggest that gp22 undergoes limited proteolytic processing which eliminates the short C-terminal piece from the molecule during the early steps of prohead assembly. Most amino acid residues of the gp22 polypeptide chain (80%) have an alpha-helical conformation and form seven peculiar alpha-helices. A model suggesting the spatial organization of gp22 is presented. Three long alpha-helices numbered 1 (1A and 1B), 3, and 5 (5A and 5B) are packed in an antiparallel fashion along the major axis of the road-shaped molecule. Two rather short alpha-helices (2 and 4) are located at the distal and proximal ends of the protein molecule, respectively. Helix number 2, which is a proteolytic fragment of gp22 found in mature T4 heads, is packed with helices 1A and 3, similar to a novel element of supersecondary structure, the alpha alpha-corner. Helix number 4 probably interacts with the gp20 connector of the prohead. The implications of the structure of the gp22 molecule for the assembly of the prohead core are discussed.
Journal of Structural Biology 104(1-3):24-31. · 3.41 Impact Factor
