Chambert R, Benyahia F, Petit-Glatron M F
Institut Jacques Monod, Université Paris, France.
Biochem J. 1990 Jan 15;265(2):375-82. doi: 10.1042/bj2650375.
The refolding of levansucrase denatured by urea was studied as a possible model for the second step of the secretion pathway of this protein. The folding-unfolding transition was monitored by measuring intrinsic fluorescence and resistance to proteolysis. Both methods provided the same estimation for the unfolding free energy of levansucrase, delta GD, which was 30.1 +/- 1.7 kJ.mol-1 (7.2 +/- 0.4 kcal.mol-1) at pH 7 in 0.1 M-potassium phosphate buffer. The rate of refolding was greatly enhanced by Fe3+, whereas the Fe3+ chelator EDTA prevented correct refolding. Fe3+ allowed the protein to reach its folded form in medium in which the dielectric constant had been lowered by ethanol. The efficiency in vivo of the export of levansucrase bearing an amino acid modification which blocks the second step of the translocation pathway was greatly increased by high concentrations of Fe3+ in the culture medium. Assuming that the protein folding governs the second step of the secretion process of levansucrase, we discuss from an irreversible thermodynamic point of view the possible role of Fe3+ in the efficient coupling of the two events.
研究了由尿素变性的蔗糖酶的重折叠，以此作为该蛋白质分泌途径第二步的可能模型。通过测量内在荧光和对蛋白酶解的抗性来监测折叠-去折叠转变。两种方法对蔗糖酶的去折叠自由能ΔGD给出了相同的估计值，在pH 7的0.1 M磷酸钾缓冲液中，该值为30.1±1.7 kJ·mol-1（7.2±0.4 kcal·mol-1）。Fe3+大大提高了重折叠速率，而Fe3+螯合剂EDTA则阻止了正确的重折叠。Fe3+能使蛋白质在乙醇降低了介电常数的介质中达到其折叠形式。培养基中高浓度的Fe3+极大地提高了带有阻止转运途径第二步的氨基酸修饰的蔗糖酶的体内输出效率。假设蛋白质折叠控制着蔗糖酶分泌过程的第二步，我们从不可逆热力学的角度讨论了Fe3+在这两个事件有效偶联中可能的作用。
