Colomer-Pallas Anne, Pereira Yannick, Petit-Glatron Marie-Françoise, Chambert Régis
Institut Jacques Monod, Laboratoire Génétique et Membranes, Centre National de la Recherche Scientifique, Universités Paris 6 et Paris 7, Tour 43, 2 place Jussieu, 75251 Paris cedex 05, France.
Biochem J. 2003 Feb 1;369(Pt 3):731-8. doi: 10.1042/BJ20021459.
We characterized the reversible folding-unfolding transition of Bacillus subtilis exocellular chitosanase from either thermal or urea denaturation of the protein. The transitions were monitored in each case by intrinsic fluorescence changes and resistance to proteolysis. Unfolding and refolding kinetics and differential scanning calorimetry analysis suggested a two-state equilibrium. The equilibrium between the folded and unfolded states was rapidly displaced towards the folded state in the presence of a low concentration of calcium (2-20 mM). The binding titration curve indicated that chitosanase possesses one weak Ca(2+)-binding site (with an equilibrium affinity constant, K (A), of 0.3x10(3) M(-1)). These results support the hypothesis that this metal ion, which is accumulated in the cell wall environment of B. subtilis, is an effector that influences folding and stability of newly translocated proteins.
我们通过枯草芽孢杆菌胞外壳聚糖酶的热变性或尿素变性来表征其可逆的折叠-去折叠转变过程。每种情况下，均通过内在荧光变化和对蛋白酶解的抗性来监测转变过程。去折叠和再折叠动力学以及差示扫描量热法分析表明存在双态平衡。在低浓度钙（2 - 20 mM）存在的情况下，折叠态与去折叠态之间的平衡迅速向折叠态移动。结合滴定曲线表明壳聚糖酶具有一个弱的Ca(2+)结合位点（平衡亲和常数K(A)为0.3×10(3) M(-1)）。这些结果支持了这样的假设，即这种在枯草芽孢杆菌细胞壁环境中积累的金属离子是一种影响新转运蛋白折叠和稳定性的效应物。
