The effect of core destabilization on the mechanical resistance of I27
The effect of core destabilization on the mechanical resistance of I27
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DOI：
10.1016/s0006-3495(02)75182-5
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发表时间：
2002-07-01
影响因子：
3.4
通讯作者：
Radford, SE
中科院分区：
文献类型：
--
作者：
Brockwell, DJ;Beddard, GS;Radford, SE
目前尚不清楚机械展开是否与化学变性探索相同的途径。为了解决这一点，我们构建了五个突变127结构域的连接体（表示为（127）(5)*），并将其用于机械展开研究。该蛋白由突变体C47S、C63S 127的四个拷贝和C63S 127的一个拷贝组成。这些突变严重破坏了127的稳定性（C63S 127和C47S、C63S 127的DeltaDeltaG（UN）分别为8.7和17.9 kJ mol(-1)）。这两个突变维持了A'和G链之间的氢键网络，这被认为是127的机械阻力的主要区域。使用原子力显微镜测量展开（127）(5)*所需的力的速度依赖性，可以可靠地评估蛋白质的内在展开速率常数(2.0 x 10（-3) s(-1)）。通过化学变性测量的展开速率常数要快五倍以上(1.1 x 10(-2) s(-1))，这表明这些技术探测了不同的展开途径。此外，通过比较野生型127串联体的力学展开参数与（127）(5)*的力学展开参数，我们表明，尽管观察到的力相当低，但岩心不稳定对确定该区域的机械灵敏度几乎没有影响。
It is still unclear whether mechanical unfolding probes the same pathways as chemical denaturation. To address this point, we have constructed a concatamer of five mutant 127 domains (denoted (127)(5)*) and used it for mechanical unfolding studies. This protein consists of four copies of the mutant C47S, C63S 127 and a single copy of C63S 127. These mutations severely destabilize 127 (DeltaDeltaG(UN) = 8.7 and 17.9 kJ mol(-1) for C63S 127 and C47S, C63S 127, respectively). Both mutations maintain the hydrogen bond network between the A' and G strands postulated to be the major region of mechanical resistance for 127. Measuring the speed dependence of the force required to unfold (127)(5)* in triplicate using the atomic force microscope allowed a reliable assessment of the intrinsic unfolding rate constant of the protein to be obtained (2.0 x 10(-3) s(-1)). The rate constant of unfolding measured by chemical denaturation is over fivefold faster (1.1 x 10(-2) s(-1)), suggesting that these techniques probe different unfolding pathways. Also, by comparing the parameters obtained from the mechanical unfolding of a wild-type 127 concatamer with that of (127)(5)*, we show that although the observed forces are considerably lower, core destabilization has little effect on determining the mechanical sensitivity of this domain.