Perry K M, Onuffer J J, Gittelman M S, Barmat L, Matthews C R
Department of Chemistry, Pennsylvania State University, University Park 16802.
Biochemistry. 1989 Sep 19;28(19):7961-8. doi: 10.1021/bi00445a061.
To test the possibility that long-range interactions might influence the folding and stability of dihydrofolate reductase, a series of single and double mutations at positions 28 and 139 were constructed and their urea-induced unfolding reactions studied by absorbance and circular dichroism spectroscopy. The alpha carbons of the two side chains are separated by 15 A in the native conformation. The replacement of Leu 28 by Arg and of Glu 139 by Gln resulted in additive effects on both kinetic and equilibrium properties of the reversible unfolding transition; no evidence for interaction was obtained. In contrast, the Arg 28/Lys 139 double replacement changed the equilibrium folding model from two state to multistate and showed evidence for interaction in one of the two kinetic phases detected in both unfolding and refolding reactions. The results can be explained in terms of a long-range, repulsive electrostatic interaction between the cationic side chains at these two positions.
为了测试长程相互作用可能影响二氢叶酸还原酶折叠和稳定性的可能性，构建了一系列位于28位和139位的单突变和双突变体，并通过吸光度和圆二色光谱研究了它们的尿素诱导的去折叠反应。在天然构象中，这两个侧链的α碳原子相距15埃。用精氨酸取代28位的亮氨酸以及用谷氨酰胺取代139位的谷氨酸，对可逆去折叠转变的动力学和平衡性质产生了累加效应；未获得相互作用的证据。相比之下，28位的精氨酸/139位的赖氨酸双取代将平衡折叠模型从两态转变为多态，并且在去折叠和重折叠反应中检测到的两个动力学阶段之一显示出相互作用的证据。这些结果可以用这两个位置的阳离子侧链之间的长程排斥静电相互作用来解释。
