Lord M, Magnin T, Yudkin M D
Microbiology Unit, Department of Biochemistry, University of Oxford, United Kingdom.
J Bacteriol. 1996 Dec;178(23):6730-5. doi: 10.1128/jb.178.23.6730-6735.1996.
We have studied the ability of three mutant forms of SpoIIAA, containing amino acid substitutions at the site of phosphorylation (serine 58), to interact with SpoIIAB. Native gel analysis revealed that SpoIIAAS58A could form a complex with SpoIIAB in the presence of ADP and more strongly in the presence of ATP. SpoIIAAS58N did not form a complex with SpoIIAB in the presence of ADP but displayed some interaction with SpoIIAB in the presence of ATP. SpoIIAAS58D was unable to form a complex with SpoIIAB in the presence of either ADP or ATP. Corresponding differences were found in the behavior of the three mutant proteins when studied by gel permeation with high-performance liquid chromatography and limited proteolysis. SpoIIAAS58A behaved like the wild-type SpoIIAA, SpoIIAAS58D like SpoIIAA-P, and SpoIIAAS58N in a way that was intermediate between the behaviors of SpoIIAA and SpoIIAA-P. Limited proteolysis was also used to show that on binding of ADP or ATP SpoIIAB undergoes a shift in conformation. The affinity of SpoIIAB for ADP and ATP was determined by limited proteolysis in the presence of a wide range of nucleotide concentrations. The results indicated that SpoIIAB has approximately equal affinity for ADP and for ATP.
我们研究了三种SpoIIAA突变形式与SpoIIAB相互作用的能力，这些突变形式在磷酸化位点（丝氨酸58）存在氨基酸替换。天然凝胶分析显示，SpoIIAAS58A在ADP存在下可与SpoIIAB形成复合物，在ATP存在下形成的复合物更强。SpoIIAAS58N在ADP存在下不与SpoIIAB形成复合物，但在ATP存在下与SpoIIAB有一些相互作用。SpoIIAAS58D在ADP或ATP存在下均无法与SpoIIAB形成复合物。在用高效液相色谱进行凝胶渗透和有限蛋白酶解研究时，发现这三种突变蛋白的行为存在相应差异。SpoIIAAS58A的行为类似于野生型SpoIIAA，SpoIIAAS58D类似于SpoIIAA-P，而SpoIIAAS58N的行为介于SpoIIAA和SpoIIAA-P之间。有限蛋白酶解还用于表明，在结合ADP或ATP时，SpoIIAB的构象会发生变化。通过在广泛的核苷酸浓度存在下进行有限蛋白酶解，测定了SpoIIAB对ADP和ATP的亲和力。结果表明，SpoIIAB对ADP和ATP的亲和力大致相等。
