| Autor: |
Efimov VP; Ivanovsky Institute of Virology, Moscow, Russia., Nepluev IV, Sobolev BN, Zurabishvili TG, Schulthess T, Lustig A, Engel J, Haener M, Aebi U, Venyaminov SYu, et. al. |
| Jazyk: |
angličtina |
| Zdroj: |
Journal of molecular biology [J Mol Biol] 1994 Sep 30; Vol. 242 (4), pp. 470-86. |
| DOI: |
10.1006/jmbi.1994.1595 |
| Abstrakt: |
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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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