Towards deconvoluting the interaction of the Bacillus subtilis sporulation proteins SinR and SinI using tryptophan analogue incorporated proteins
Sporulation in Bacillus subtilis is used as the strategy of last resort for survival of the organism and it is a very tightly controlled developmental process. One of the control checkpoints that must be overcome for sporulation to occur is the repression of sporulation genes by the protein SinR (13.5 kDa). This is done by binding of the anti-repressor SinI (6.5 kDa) to form a tightly bound heterodimer. To investigate the interaction of SinI with SinR in solution, an analytical ultracentrifuge study was undertaken. SinR was found to be a tetramer, whereas SinI was in a monomer/dimer equilibrium. Derivatives of both SinI and SinR, where the native tryptophan was replaced by the analogue 7-aza-tryptophan (7AW), were expressed and found to be as active as the wild-type proteins. The 7AW proteins have the property of having significant absorbance at 315 nm, thus allowing them to be monitored even in the presence of tryptophan containing proteins, making them ideal for studying protein/protein interactions.
Key wordsSporulation proteins Bacillus subtilis Tryptophan 7-Azatryptophan Analytical ultracentrifugation
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