Jelesarov I, Dürr E, Thomas R M, Bosshard H R
Biochemisches Institut der Universität Zürich, Switzerland.
Biochemistry. 1998 May 19;37(20):7539-50. doi: 10.1021/bi972977v.
The stability of a coiled coil or leucine zipper is controlled by hydrophobic interactions and electrostatic forces between the constituent helices. We have designed a 30-residue peptide with the repeating seven-residue pattern of a coiled coil, (abcdefg)n, and with Glu in positions e and g of each heptad. The glutamate side chains prevented folding at pH values above 6 because of electrostatic repulsion across the helix dimer interface as well as within the individual helices. Protonation of the carboxylates changed the conformation from a random coil monomer to a coiled coil dimer. Folding at alkaline pH where the peptide had a net charge of -7e was promoted by the addition of salts. The nature of the charge screening cation was less important than that of the anion. The high salt concentrations (>1 M) necessary to induce folding indicated that the salt-induced folding resulted from alterations in the protein-water interaction. Folding was promoted by the kosmotropic anions sulfate and fluoride and to a lesser extent by the weak kosmotrope formate, whereas chloride and the strong chaotrope perchlorate were ineffective. Kosmotropes are excluded from the protein surface, which is preferentially hydrated, and this promotes folding by strengthening hydrophobic interactions at the coiled coil interface. Although charge neutralization also contributed to folding, it was effective only when the screening cation was partnered by a good kosmotropic anion. Folding conformed to a two-state transition from random coil monomer to coiled coil dimer and was enthalpy driven and characterized by a change in the heat capacity of unfolding of 3.9 +/- 1.2 kJ mol-1 K-1. The rate of folding was analyzed by fluorescence stopped-flow measurements. Folding occurred in a biphasic reaction in which the rapid formation of an initial dimer (kf = 2 x 10(7) M-1 s-1) was followed by an equally rapid concentration-independent rearrangement to the folded dimer (k > 100 s-1).
卷曲螺旋或亮氨酸拉链的稳定性由组成螺旋之间的疏水相互作用和静电力控制。我们设计了一种30个残基的肽，其具有卷曲螺旋重复的七残基模式(abcdefg)n，且在每个七肽的e和g位置为Glu。由于跨螺旋二聚体界面以及单个螺旋内的静电排斥，谷氨酸侧链在pH值高于6时阻止折叠。羧酸盐的质子化使构象从无规卷曲单体转变为卷曲螺旋二聚体。在碱性pH下（此时肽的净电荷为-7e），添加盐可促进折叠。电荷屏蔽阳离子的性质不如阴离子重要。诱导折叠所需的高盐浓度(>1 M)表明盐诱导的折叠是由蛋白质-水相互作用的改变引起的。促溶剂阴离子硫酸根和氟离子可促进折叠，弱促溶剂甲酸根的促进作用较小，而氯离子和强离液剂高氯酸根则无效。促溶剂被排除在优先水合的蛋白质表面之外，这通过加强卷曲螺旋界面处的疏水相互作用来促进折叠。虽然电荷中和也有助于折叠，但只有当屏蔽阳离子与良好的促溶剂阴离子配对时才有效。折叠符合从无规卷曲单体到卷曲螺旋二聚体的两态转变，由焓驱动，其特征是解折叠热容量变化为3.9±1.2 kJ mol-1 K-1。通过荧光停流测量分析折叠速率。折叠发生在双相反应中，其中首先快速形成初始二聚体(kf = 2 x 10(7) M-1 s-1)，随后同样快速地进行与浓度无关的重排形成折叠二聚体(k > 100 s-1)。
