Huang Fang, Settanni Giovanni, Fersht Alan R
Medical Research Council Centre for Protein Engineering and Cambridge University Chemical Laboratory, Hills Road, Cambridge CB2 0QH, UK.
Protein Eng Des Sel. 2008 Mar;21(3):131-46. doi: 10.1093/protein/gzm069. Epub 2008 Jan 18.
The Engrailed Homeodomain folds on the microsecond time scale via an intermediate that is experimentally well characterised using structural Engrailed-Homeodomain mimics. Here, we analysed directly the changes in distance between key residues during the kinetics of unfolding and at equilibrium using fluorescence resonance energy transfer (FRET). Trp was the donor and 5-(((acetylamino)ethyl)amino) naphthalene-1-sulphate, the acceptor, substituted in positions that caused little change in stability. Distances calculated for the native state were in good agreement with those derived from the NMR structure. The distances between the N- and C-termini of Helix I and of Helix III increased, then decreased and finally increased again with increasing GdmCl concentration on equilibrium denaturation. This behaviour implied that there was a folding intermediate on the folding pathway and that this intermediate was populated at low concentrations of GdmCl concentration ( approximately 1 M). We analysed the changes in distance during temperature-jump relaxation kinetics, using a qualitative and very conservative procedure that drew conclusions only when changes in fluorescence of mutants containing either the donor or the acceptor alone would not obscure the change in the FRET signal when both donor and acceptor were present. The distance changes obtained under equilibrium and kinetic measurements were self-consistent and also consistent with the known high-resolution structures of the mimics of the folding intermediates. We showed that for analysing distances in disordered ensembles, it is important to use FRET probes with a critical distance close to the average separation in the ensemble. Otherwise, average distances could be over or underestimated.
Engrailed 同源结构域在微秒时间尺度上通过一个中间体折叠，该中间体可通过使用结构 Engrailed 同源结构域模拟物进行实验很好地表征。在这里，我们使用荧光共振能量转移（FRET）直接分析了在展开动力学过程中和平衡时关键残基之间距离的变化。色氨酸作为供体，5 - （（（乙酰氨基）乙基）氨基）萘 - 1 - 硫酸盐作为受体，取代在对稳定性影响很小的位置。计算得到的天然状态下的距离与从 NMR 结构推导的距离非常吻合。在平衡变性时，随着 GdmCl 浓度增加，螺旋 I 和螺旋 III 的 N 端和 C 端之间的距离先增加，然后减小，最后再次增加。这种行为表明在折叠途径上存在一个折叠中间体，并且这个中间体在低浓度 GdmCl（约 1 M）时出现。我们使用一种定性且非常保守的程序分析了温度跳跃弛豫动力学过程中的距离变化，该程序仅在仅含有供体或受体的突变体的荧光变化不会掩盖同时存在供体和受体时 FRET 信号的变化时才得出结论。在平衡和动力学测量下获得的距离变化是自洽的，并且也与折叠中间体模拟物的已知高分辨率结构一致。我们表明，对于分析无序集合中的距离，使用临界距离接近集合中平均间距的 FRET 探针很重要。否则，平均距离可能会被高估或低估。
