Wendt H, Leder L, Härmä H, Jelesarov I, Baici A, Bosshard H R
Biochemisches Institut der Universität Zürich, Switzerland.
Biochemistry. 1997 Jan 7;36(1):204-13. doi: 10.1021/bi961672y.
Leucine zippers (coiled coils) are dimerization motifs found in several DNA-binding transcription factors. A parallel leucine zipper composed of the acidic chain X1-EYQALEKEVAQLEAENX2-ALEKEVAQLEHEG-amide and the basic chain X1-EYQALKKKVAQLKAKNX2ALKKKVAQLKHKG-amide was designed to study the kinetics of folding of a heterodimeric leucine zipper and to investigate the role of electrostatic attraction between oppositely charged peptide chains to the folding reaction. Each peptide alone did not form a leucine zipper at ionic strength (mu) < 1 M because of electrostatic repulsion between like charges in a homodimer. Therefore, the formation of the heterodimeric leucine zipper could be investigated by simple mixing of acidic and basic chains. To monitor folding, a fluorescent label was located either at the N-terminus (X1 = fluorescein-GGG, X2 = Q) or in the center of the coiled coil (X1 = acetyl, X2 = W). Folding could be described by a simple two-state reaction involving the disordered monomers and the folded heterodimer. The same bimolecular rate constant (k(on)) was observed independent of the location of the fluorescent label, indicating that both fluorescence probes monitored the same reaction. Lowering of the ionic strength increased k(on) from 4 x 10(6) M-1 s-1 (mu = 525 mM) to about 5 x 10(7) M-1 s-1 (mu = 74 mM). When extrapolated to mu = O, k(on) was approximately 10(9) M-1 s-1, which is near the diffusion limit. In contrast, the rate of dissociation depended very weakly on ionic strength; k(off) decreased only by about 2-fold when mu was lowered from 525 to 74 mM. Equilibrium association constants (Ka) of the heterodimeric zippers measured directly and calculated from kinetic constants (Ka = k(on)/k(off) were in good agreement. The observed two-state mechanism, the strong dependence on ionic strength of k(on) but not of k(off), and the nearly diffusion-limited association rate at very low ionic strength point to a folding pathway in which the formation of an electrostatically stabilized dimeric intermediate may be rate-limiting and the subsequent folding to the final dimer is very rapid and follows a "down-hill" free energy landscape. The small increase of k(off) at increasing ionic strength indicates a minor contribution of electrostatics to the stability of the folded leucine zipper.
亮氨酸拉链（卷曲螺旋）是在几种DNA结合转录因子中发现的二聚化基序。设计了一种由酸性链X1-EYQALEKEVAQLEAENX2-ALEKEVAQLEHEG-酰胺和碱性链X1-EYQALKKKVAQLKAKNX2ALKKKVAQLKHKG-酰胺组成的平行亮氨酸拉链，以研究异二聚体亮氨酸拉链的折叠动力学，并研究带相反电荷的肽链之间的静电引力对折叠反应的作用。由于同二聚体中相同电荷之间的静电排斥，在离子强度（μ）<1 M时，每种肽单独都不会形成亮氨酸拉链。因此，可以通过简单混合酸性链和碱性链来研究异二聚体亮氨酸拉链的形成。为了监测折叠，荧光标记位于N端（X1 = 荧光素-GGG，X2 = Q）或卷曲螺旋的中心（X1 = 乙酰基，X2 = W）。折叠可以通过一个简单的两态反应来描述，该反应涉及无序单体和折叠的异二聚体。观察到相同的双分子速率常数（k(on)），与荧光标记的位置无关，这表明两种荧光探针监测的是相同的反应。降低离子强度会使k(on)从4×10^6 M^-1 s^-1（μ = 525 mM）增加到约5×10^7 M^-1 s^-1（μ = 74 mM）。外推到μ = 0时，k(on)约为10^9 M^-1 s^-1，接近扩散极限。相比之下，解离速率对离子强度的依赖性非常弱；当μ从525 mM降低到74 mM时，k(off)仅降低约2倍。直接测量的和根据动力学常数计算的（Ka = k(on)/k(off)）异二聚体拉链的平衡缔合常数（Ka）吻合良好。观察到的两态机制、k(on)对离子强度的强烈依赖性而非k(off)对离子强度的强烈依赖性，以及在非常低的离子强度下接近扩散极限的缔合速率，表明存在一种折叠途径，其中静电稳定的二聚体中间体的形成可能是限速步骤，随后折叠成最终二聚体非常迅速，并遵循“下坡”自由能态势。随着离子强度增加，k(off)的小幅增加表明静电对折叠后的亮氨酸拉链稳定性的贡献较小。
