Missiakas D, Betton J M, Chaffotte A, Minard P, Yon J M
Laboratoire d'Enzymologie Physicochimique et Moléculaire, Unité de Recherche du Centre National de la Recherche Scientifique, Université de Paris-Sud, Orsay, France.
Protein Sci. 1992 Nov;1(11):1485-93. doi: 10.1002/pro.5560011110.
Unfolding and refolding kinetics of yeast phosphoglycerate kinase were studied by following the time-dependent changes of two signals: the ellipticity at 218 nm and 222 nm, and the fluorescence emission at 330 nm (following excitation at 295 nm). The protein is composed of two similar-sized structural domains. Each domain has been produced by recombinant DNA techniques. It has been previously demonstrated that the engineered isolated domains are able to fold into a quasinative structure (Minard, P., et al., 1989b, Protein Eng. 3, 55-60; Missiakas, D., Betton, J.M., Minard, P., & Yon, J.M., 1990, Biochemistry 29, 8683-8689). The behavior of the isolated domains was studied using the same two conformational probes as for the whole enzyme. We found that the refolding kinetics of each domain are multiphasic. In the whole protein, domain folding and pairing appeared to be simultaneous events. However, it was found that some refolding steps occurring during the refolding of the isolated C-domain are masked during the refolding of yeast phosphoglycerate kinase. The N-domain was also found to refold faster when it was isolated than when integrated.
通过跟踪两个信号随时间的变化，研究了酵母磷酸甘油酸激酶的去折叠和重折叠动力学：218nm和222nm处的椭圆率，以及330nm处的荧光发射（在295nm激发后）。该蛋白质由两个大小相似的结构域组成。每个结构域均通过重组DNA技术产生。先前已经证明，工程化的分离结构域能够折叠成准天然结构（米纳德，P.等人，1989b，《蛋白质工程》3，55 - 60；米西亚卡斯，D.，贝顿，J.M.，米纳德，P.，& 扬，J.M.，1990，《生物化学》29，8683 - 8689）。使用与整个酶相同的两种构象探针研究了分离结构域的行为。我们发现每个结构域的重折叠动力学是多相的。在整个蛋白质中，结构域折叠和配对似乎是同时发生的事件。然而，发现在分离的C结构域重折叠过程中发生的一些重折叠步骤在酵母磷酸甘油酸激酶重折叠过程中被掩盖了。还发现N结构域分离时比重组时重折叠得更快。
