Localization of two protease-sensitive regions separating distinct domains in the reovirus cell-attachment protein sigma 1.
Electron microscopy and sequence analysis have suggested the presence of distinct morphological regions within the reovirus cell attachment protein sigma 1. Kinking of purified sigma 1 observed by electron microscopy and sequence-predicted flexibility profiles suggest the presence of potential flexible regions in the molecule, most notably near the N-terminus, in the neck region and near the middle of the fiber. We have mapped the trypsin and chymotrypsin cleavage sites in sigma 1 by direct amino acid sequencing of gel-purified, proteolytic fragments of purified baculovirus-expressed sigma 1. The results indicated that both proteases cleave sigma 1 several times in one, or both, of two specific regions in the molecule. Further analysis using proteases with different cleavage specificities revealed the same general digestion pattern. The two protease-sensitive regions of sigma 1 were localized to the proposed N-terminal hinge region separating the hydrophobic anchor from the coiled-coil and to the C-proximal portion of the neck separating most of the fibrous tail from the globular head. The protease susceptibility of these regions indicates an open, accessible conformation supporting the notion of flexible regions that may be important in sigma 1 function.
Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada.
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Duncan
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Lee
D000595:Amino Acid Sequence; D001665:Binding Sites; D036022:Capsid Proteins; D002918:Chymotrypsin / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012087:Reoviridae / Q000737:chemistry*; D014357:Trypsin / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
D036022:Capsid Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C031728:sigma 1 protein, reovirus; D010450:Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin