Quaternary re-arrangement analysed by spectral enhancement: the interaction of a sporulation repressor with its antagonist.
The protein/protein interaction between SinI and SinR has been studied by analytical ultracentrifugation and gel electrophoresis in an attempt to understand how these proteins contribute to developmental control of sporulation in Bacillus subtilis. SinR was found to be tetrameric, while SinI was found to exist as monomers and dimers in a rapidly reversible equilibrium. Labelling of SinR by incorporating the tryptophan analogue 7-azatryptophan (7AW) into the protein in place of tryptophan shifts the UV absorbance spectrum, thus allowing selective monitoring of 7AWSinR at 315 nm using the UV absorption optics of the analytical ultracentrifuge. Selective monitoring of SinR in mixtures of SinR and SinI enables the binding and stoichiometry of the interaction to be investigated quantitatively and unambiguously. We demonstrate that the oligomeric forms of SinR and SinI re-arrange to form a tight 1:1 SinR:SinI complex, with no stable intermediate species. A fragment of SinR, SinR(1-69), which contains only the DNA-binding domain, was found to be monomeric, showing that the protein appears not to oligomerise in a similar manner to the Cro repressor, a protein with which it shares a marked structural similarity.
Scott
D J
DJ
Department of Biology, University of York, Heslington, UK.
Leejeerajumnean
S
S
Brannigan
J A
JA
Lewis
R J
RJ
Wilkinson
A J
AJ
Hoggett
J G
JG
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000737:chemistry* / Q000502:physiology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013171:Spores, Bacterial / Q000737:chemistry / Q000502:physiology; D014364:Tryptophan / Q000031:analogs & derivatives / Q000378:metabolism; D014461:Ultracentrifugation
D001426:Bacterial Proteins; D012097:Repressor Proteins; C097712:SinI protein, Bacillus subtilis; C076733:FlaD protein, Bacteria; D014364:Tryptophan