Holtzer M E, Bretthorst G L, d'Avignon D A, Angeletti R H, Mints L, Holtzer A
Department of Chemistry, Washington University, St. Louis, Missouri 63130-4899, USA.
Biophys J. 2001 Feb;80(2):939-51. doi: 10.1016/s0006-3495(01)76073-0.
Studies by one-dimensional NMR are reported on the interconversion of folded and unfolded forms of the GCN4 leucine zipper in neutral saline buffer. The peptide bears 99% 13C(alpha) labels at three sites: V9, L12, and G31. Time-domain 13C(alpha)-NMR spectra are interpreted by global Bayesian lineshape analysis to extract the rate constants for both unfolding and folding as functions of temperature in the range 47-71 degrees C. The data are well fit by the assumption that the same rate constants apply at each labeled site, confirming that only two conformational states need be considered. Results show that 1) both processes require a free energy of activation; 2) unfolding is kinetically enthalpy-opposed and entropy-driven, while folding is the opposite; and 3) the transition state dimer ensemble averages approximately 40% helical. The activation parameters for unfolding, derived from NMR data at the elevated temperatures where both conformations are populated, lead to estimates of the rate constant at low temperatures (5-15 degrees C) that agree with extant values determined by stopped-flow CD via dilution from denaturing media. However, the corresponding estimated values for the folding rate constant are larger by two to three orders of magnitude than those obtained by stopped flow. We propose that this apparent disagreement is caused by the necessity, in the stopped-flow experiment, for initiation of new helices as the highly denaturant-unfolded molecule adjusts to the newly created benign solvent conditions. This must reduce the success rate of collisions in producing the folded molecule. In the NMR determinations, however, the unfolded chains always have a small, but essential, helix content that makes such initiation unnecessary. Support for this hypothesis is adduced from recent extant experiments on the helix-coil transition in single-chain helical peptides and from demonstration that the folding rate constants for coiled coils, as obtained by stopped flow, are influenced by the nature of the denaturant used.
报道了在中性盐水缓冲液中，通过一维核磁共振研究GCN4亮氨酸拉链折叠和未折叠形式的相互转化。该肽在三个位点带有99%的13C（α）标记：V9、L12和G31。通过全局贝叶斯线形分析解释时域13C（α）-核磁共振谱，以提取47-71摄氏度范围内作为温度函数的解折叠和折叠速率常数。假设每个标记位点适用相同的速率常数，数据拟合良好，证实只需考虑两种构象状态。结果表明：1）两个过程都需要活化自由能；2）解折叠在动力学上是焓对抗和熵驱动的，而折叠则相反；3）过渡态二聚体集合平均约40%为螺旋结构。在两种构象都存在的高温下，从核磁共振数据得出的解折叠活化参数，导致低温（5-15摄氏度）下速率常数的估计值与通过从变性介质稀释的停流圆二色法测定的现有值一致。然而，折叠速率常数的相应估计值比通过停流获得的值大两到三个数量级。我们认为，这种明显的差异是由于在停流实验中，当高度变性的未折叠分子适应新创建的良性溶剂条件时，需要启动新的螺旋结构。这必然会降低产生折叠分子时碰撞的成功率。然而，在核磁共振测定中，未折叠链总是有少量但必不可少的螺旋含量，使得这种启动没有必要。最近关于单链螺旋肽中螺旋-线圈转变的现有实验以及证明通过停流获得的卷曲螺旋折叠速率常数受所用变性剂性质影响，都支持了这一假设。
