Du Deguo, Tucker Matthew J, Gai Feng
Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Biochemistry. 2006 Feb 28;45(8):2668-78. doi: 10.1021/bi052039s.
The folding kinetics of a 16-residue beta-hairpin (trpzip4) and five mutants were studied by a laser-induced temperature-jump infrared method. Our results indicate that mutations which affect the strength of the hydrophobic cluster lead to a decrease in the thermal stability of the beta-hairpin, as a result of increased unfolding rates. For example, the W45Y mutant has a phi-value of approximately zero, implying a folding transition state in which the native contacts involving Trp45 are not yet formed. On the other hand, mutations in the turn or loop region mostly affect the folding rate. In particular, replacing Asp46 with Ala leads to a decrease in the folding rate by roughly 9 times. Accordingly, the phi-value for D46A is determined to be approximately 0.77, suggesting that this residue plays a key role in stabilizing the folding transition state. This is most likely due to the fact that the main chain and side chain of Asp46 form a characteristic hydrogen bond network with other residues in the turn region. Taken together, these results support the folding mechanism we proposed before, which suggests that the turn formation is the rate-limiting step in beta-hairpin folding and, consequently, a stronger turn-promoting sequence increases the stability of a beta-hairpin primarily by increasing its folding rate, whereas a stronger hydrophobic cluster increases the stability of a beta-hairpin primarily by decreasing its unfolding rate. In addition, we have examined the compactness of the thermally denatured and urea-denatured states of another 16-residue beta-hairpin, using the method of fluorescence resonance energy transfer. Our results show that the thermally denatured state of this beta-hairpin is significantly more compact than the urea-denatured state, suggesting that the very first step in beta-hairpin folding, when initiated from an extended conformation, probably corresponds to a process of hydrophobic collapse.
通过激光诱导温度跳跃红外方法研究了一个16残基β-发夹（trpzip4）及其五个突变体的折叠动力学。我们的结果表明，影响疏水簇强度的突变会导致β-发夹热稳定性降低，这是由于解折叠速率增加所致。例如，W45Y突变体的φ值约为零，这意味着折叠过渡态中尚未形成涉及Trp45的天然接触。另一方面，转角或环区的突变主要影响折叠速率。特别是，用丙氨酸取代天冬氨酸46会导致折叠速率降低约9倍。因此，D46A的φ值确定约为0.77，这表明该残基在稳定折叠过渡态中起关键作用。这很可能是因为天冬氨酸46的主链和侧链与转角区的其他残基形成了特征性氢键网络。综上所述，这些结果支持了我们之前提出的折叠机制，该机制表明转角形成是β-发夹折叠中的限速步骤，因此，更强的促进转角形成的序列主要通过提高其折叠速率来增加β-发夹的稳定性，而更强的疏水簇主要通过降低其解折叠速率来增加β-发夹的稳定性。此外，我们使用荧光共振能量转移方法研究了另一个16残基β-发夹的热变性和尿素变性状态的紧密程度。我们的结果表明，该β-发夹的热变性状态比尿素变性状态明显更紧密，这表明β-发夹从伸展构象开始折叠时的第一步可能对应于疏水塌缩过程。
