Muñoz Victor, Ghirlando Rodolfo, Blanco Francisco J, Jas Gouri S, Hofrichter James, Eaton William A
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA.
Biochemistry. 2006 Jun 13;45(23):7023-35. doi: 10.1021/bi052556a.
We have investigated the solution structure, equilibrium properties, and folding kinetics of a 17-residue beta-hairpin-forming peptide derived from the protein ubiquitin. NMR experiments show that at 4 degrees C the peptide has a highly populated beta-hairpin conformation. At protein concentrations higher than 0.35 mM, the peptide aggregates. Sedimentation equilibrium measurements show that the aggregate is a trimer, while NMR indicates that the beta-hairpin conformation is maintained in the trimer. The relaxation kinetics in nanosecond laser temperature-jump experiments reveal a concentration-independent microsecond phase, corresponding to beta-hairpin unfolding-refolding, and a concentration-dependent millisecond phase due to oligomerization. Kinetic modeling of the relaxation rates and amplitudes yields the folding and unfolding rates for the monomeric beta-hairpin, as well as assembly and disassembly rates for trimer formation consistent with the equilibrium constant determined by sedimentation equilibrium. When the net charge on the peptides and ionic strength were taken into account, the rate of trimer assembly approaches the Debye-Smoluchowski diffusion limit. At 300 K, the rate of formation of the monomeric hairpin is (17 micros)(-1), compared to rates of (0.8 micros)(-1) to (52 micros)(-1) found for other peptides. After using Kramers theory to correct for the temperature dependence of the pre-exponential factor, the activation energy for hairpin formation is near zero, indicating that the barrier to folding is purely entropic. Comparisons with previously measured rates for a series of hairpins are made to distinguish between zipper and hydrophobic collapse mechanisms. Overall, the experimental data are most consistent with the zipper mechanism in which structure formation is initiated at the turn, the mechanism predicted by the Ising-like statistical mechanical model that was developed to explain the equilibrium and kinetic data for the beta-hairpin from protein GB1. In contrast, the majority of simulation studies favor a hydrophobic collapse mechanism. However, with few exceptions, there is little or no quantitative comparison of the simulation results with experimental data.
我们研究了一种源自泛素蛋白的17个残基的β-发夹形成肽的溶液结构、平衡性质和折叠动力学。核磁共振实验表明，在4℃时，该肽具有高度富集的β-发夹构象。当蛋白质浓度高于0.35 mM时，该肽会聚集。沉降平衡测量表明，聚集体是三聚体，而核磁共振表明β-发夹构象在三聚体中得以维持。纳秒激光温度跳跃实验中的弛豫动力学揭示了一个与浓度无关的微秒相，对应于β-发夹的解折叠-再折叠，以及一个由于寡聚化导致的与浓度相关的毫秒相。对弛豫速率和幅度进行动力学建模，得出了单体β-发夹的折叠和解折叠速率，以及三聚体形成的组装和解组装速率，这与沉降平衡测定的平衡常数一致。当考虑到肽上的净电荷和离子强度时，三聚体组装速率接近德拜-斯莫卢霍夫斯基扩散极限。在300 K时，单体发夹的形成速率为(17微秒)(-1)，相比之下，其他肽的速率为(0.8微秒)(-1)至(52微秒)(-1)。在使用克莱默斯理论校正了预指数因子的温度依赖性后，发夹形成的活化能接近零，这表明折叠的障碍纯粹是熵性的。与之前测量的一系列发夹的速率进行比较，以区分拉链和疏水塌缩机制。总体而言，实验数据与拉链机制最为一致，在该机制中，结构形成始于转角处，这是为解释来自蛋白质GB1的β-发夹的平衡和动力学数据而开发的类伊辛统计力学模型所预测的机制。相比之下，大多数模拟研究支持疏水塌缩机制。然而，除了少数例外，模拟结果与实验数据几乎没有或没有定量比较。
