Conformational transitions within the calmodulin-binding site of Bordetella pertussis adenylate cyclase studied by time-resolved fluorescence of Trp242 and circular dichroism

被引:12
作者
Bouhss, A
Vincent, M
Munier, H
Gilles, AM
Takahashi, M
Barzu, O
Danchin, A
Gallay, J
机构
[1] UNIV PARIS 11,UTILISAT RAYONNEMENT ELECTROMAGNET LAB,CNRS,CEA,MINIST ENSEIGNEMENT SUPER RECH,F-91405 ORSAY,FRANCE
[2] INST CURIE,ETUD MUTAGENESE & CANCEROGENESE GRP,F-91405 ORSAY,FRANCE
[3] URA 1342,ORSAY,FRANCE
[4] INST PASTEUR,UNITE BIOCHIM REGULAT CELLULAIRES,F-75724 PARIS,FRANCE
[5] INST PASTEUR,UNITE REGULAT EXPRESS GENET,F-75724 PARIS,FRANCE
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1996年 / 237卷 / 03期
关键词
adenylate cyclase; Bordetella pertussis; calmodulin binding; peptide; fluorescence properties;
D O I
10.1111/j.1432-1033.1996.0619p.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The sequence situated around Trp242 in Bordetella pertussis adenylate cyclase, a bifunctional protein of 1706 amino acid residues, forms the core of the calmodulin-binding site. Peptides varying in size and in affinity for calmodulin, and preserving the same sequence around Trp242 were analyzed by time-resolved fluorescence spectroscopy. Their dynamic properties were compared to those of the catalytic domain of B. pertussis adenylate cyclase corresponding to the first 400 amino acid residues of the protein and in which the Trp69 residue was replaced by Phe. The heterogeneity of the fluorescence intensity decays of Trp242 is likely due to the existence of conformers in equilibrium as is suggested by the effect of trifluoroethanol both on the secondary structure content and the lifetime distributions. Binding to calmodulin leads to striking effects on the lifetime distribution profiles by selecting a major excited state population and therefore one major conformer. Trp242 still presents some degree of rotational freedom in the complexes. The reduction of rotational freedom is more important for the shorter peptides than for the longest one. A similar selection of one major conformer with the same lifetime was also observed for the Trp242 in the mutant protein when bound to calmodulin, as in the complexes with the peptides. We conclude that the site of interaction of B. pertussis adenylate cyclase with calmodulin has similar conformational flexibility as that evidenced in the isolated peptides. This property of the molecule allows a better adjustment of the enzyme upon interaction with calmodulin.
引用
收藏
页码:619 / 628
页数:10
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