Brockwell David J, Beddard Godfrey S, Clarkson John, Zinober Rebecca C, Blake Anthony W, Trinick John, Olmsted Peter D, Smith D Alastair, Radford Sheena E
School of Biochemistry and Molecular Biology, University of Leeds, United Kingdom.
Biophys J. 2002 Jul;83(1):458-72. doi: 10.1016/S0006-3495(02)75182-5.
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 I27 domains (denoted (I27)(5)) and used it for mechanical unfolding studies. This protein consists of four copies of the mutant C47S, C63S I27 and a single copy of C63S I27. These mutations severely destabilize I27 (DeltaDeltaG(UN) = 8.7 and 17.9 kJ mol(-1) for C63S I27 and C47S, C63S I27, respectively). Both mutations maintain the hydrogen bond network between the A' and G strands postulated to be the major region of mechanical resistance for I27. Measuring the speed dependence of the force required to unfold (I27)(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 I27 concatamer with that of (I27)(5)*, we show that although the observed forces are considerably lower, core destabilization has little effect on determining the mechanical sensitivity of this domain.
目前尚不清楚机械展开与化学变性所探测的是否为相同途径。为解决这一问题，我们构建了一个由五个突变I27结构域组成的串联体（记为(I27)(5)*），并将其用于机械展开研究。该蛋白质由四个突变型C47S、C63S I27拷贝和一个C63S I27拷贝组成。这些突变严重破坏了I27的稳定性（C63S I27和C47S、C63S I27的ΔΔG(UN)分别为8.7和17.9 kJ mol(-1)）。两种突变都维持了A'链和G链之间的氢键网络，该网络被认为是I27机械阻力的主要区域。使用原子力显微镜对展开(I27)(5)*所需力的速度依赖性进行三次测量，从而可靠地评估了该蛋白质的固有展开速率常数（2.0×10(-3) s(-1)）。通过化学变性测量的展开速率常数快了五倍多（1.1×10(-2) s(-1)），这表明这些技术探测的是不同的展开途径。此外，通过比较从野生型I27串联体的机械展开与(I27)(5)*的机械展开中获得的参数，我们表明，尽管观察到的力要低得多，但核心去稳定化对确定该结构域的机械敏感性影响不大。
