Biochemical and biophysical characterization of the reovirus cell attachment protein sigma 1: evidence that it is a homotrimer.
The oligomerization state of the reovirus cell attachment protein sigma 1 (49K monomeric molecular weight) was determined by biochemical and biophysical means. Full-length (protein product designated A) and C-terminal truncated (protein product designated B) serotype 3 reovirus S1 mRNA transcripts synthesized in vitro were cotranslated in a rabbit reticulocyte lysate, and the products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under conditions which allowed for the identification of oligomeric forms of sigma 1. A total of four oligomeric protein bands (corresponding to A3, A2B1, A1B2, and B3, respectively) was consistently observed, which suggests that the protein is made up of three monomeric subunits. Biophysical characterization of purified sigma 1 using column filtration and sucrose gradient sedimentation analysis confirmed the highly asymmetric shape of sigma 1 and allowed us to determine the molecular weight of the native protein to be approximately 132K (a trimer). Similar biophysical analysis on the two tryptic fragments of the sigma 1 [N-terminal fibrous tail (26K monomeric molecular weight) and the C-terminal globular head (23K monomeric molecular weight)] yielded molecular weights of 77K and 64K, respectively, both again corresponding to trimers. We therefore conclude that protein sigma 1 is a homotrimer and provide, with supportive experimental evidence, a rationale for the anomalous behavior of the oligomeric protein in SDS-polyacrylamide gels, which, coupled with chemical cross-linking studies, has in part led to the previous suggestion that sigma 1 might be a higher order oligomer.
Strong
J E
JE
Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Centre, Alberta, Canada.
Leone
G
G
Duncan
R
R
Sharma
R K
RK
Lee
P W
PW
D036022:Capsid Proteins; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002872:Chromosome Deletion; D004591:Electrophoresis, Polyacrylamide Gel; D005814:Genes, Viral; D046911:Macromolecular Substances; D012089:Mammalian orthoreovirus 3 / Q000235:genetics / Q000502:physiology*; D008961:Models, Structural; D008970:Molecular Weight; D016296:Mutagenesis; D010446:Peptide Fragments / Q000302:isolation & purification; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D036022:Capsid Proteins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D012333:RNA, Messenger; D014764:Viral Proteins; C031728:sigma 1 protein, reovirus