The effect of core destabilization on the mechanical resistance of I27

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.