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Folding and aggregation kinetics of a beta-hairpin

机译：β发夹的折叠和聚合动力学

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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 mu s)(-1), compared to rates of ( 0.8 mu s)(-1) to ( 52 mu s)(-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.
机译：我们研究了衍生自蛋白质泛素的17-残基β发夹形成肽的溶液结构，平衡性能和折叠动力学。 NMR实验表明，在4摄氏度下，肽具有高度填充的β-发夹构象。在高于0.35mm的蛋白质浓度下，肽聚集体。沉积平衡测量表明，聚集体是三聚体，而NMR表明β-发夹构象保持在三聚体中。纳秒激光温度跳跃实验中的弛豫动力学显示出浓度无关的微秒阶段，对应于β-发夹展开 - 重折叠，以及由于寡聚化引起的浓度依赖性毫秒阶段。弛豫率和振荡的动力学建模产生单体β-发夹的折叠和展开速率，以及用于三聚体形成的组装和拆卸速率，其与通过沉降平衡确定​​的平衡常数一致。考虑到肽和离子强度的净电荷时，三聚集组件的速率接近Deye-Smoluchowski扩散极限。在300K时，单体发夹的形成速率是（17μs）（ - 1），与其他肽的（52μs）（ - 1）的速率相比。在使用KRamers理论对预指数因子的温度依赖性校正后，发夹形成的激活能量接近零，表明折叠屏障纯粹是熵的。对先前测量的一系列发夹的测量率进行了比较，以区分拉链和疏水塌陷机制。总的来说，实验数据与转弯启动结构形成的拉链机构最符合的是，由待解释来自蛋白质的β-发夹的平衡和动力学数据来预测的机制GB1。相比之下，大多数仿真研究有利于疏水塌陷机制。然而，少数例外情况下，使用实验数据几乎没有或没有定量比较仿真结果。

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