Mutational analysis is a high-resolution tool that permits minimal perturbations of the system to be introduced in order to interrogate the process under study with a view to revealing molecular details of the underlying mechanism. It has provided a unique way of studying the molecular basis of how proteins respond to mechanical force. Several types of mutant proteins of the titin 127 domain (Sect. 8.4.1) have been used in SMFS studies. Loop insertion using glycine residues was used to show the existence of a "mechanical clamp" in 127 (a model system in these pulling experiments, Sect. 8.4.1) and demonstrated the amino acid resolution of the technique (Carrion-Vazquez et al. 1999b). Proline mutagenesis was used to show the existence of a mechanical folding intermediate (Marszalek et al. 1999b) and to alter the mechanical stability of the domain (Li et al. 2000a). ttis approach takes advantage of the fact that proline (in addition to being a breaker of secondary structure) is an imino acid and, as such, cannot form backbone hydrogen bonds. Conservative substitution and deletion mutagenesis have been used to demonstrate the existence of the 127 folding intermediate (Fowler et al. 2002). Furthermore, analysis of the so-called mechanical ® value holds the promise for a more detailed examination of the structure of the transition state in a forced unfolding reaction (Best et al. 2002). ttis is the mechanical equivalent to the well-established O-value analysis (Fersht et al. 1992), which is used to examine the conformational effect of a mutation based on the relative changes in the free energy of the native, transition, and denatured states (AAGn-j, AAGu-t). tte ® value is obtained from the changes in thermodynamic stability measured from the shifts in the equilibrium denaturation curves, tte unfolding ® value, defined as «P^AAGnVAAGn-d. is determined by comparing the effect of the mutation on the transition state and measures the amount of native structure that is present around the mutated residue in the transition state. AAGn-d is determined using equilibrium denaturation experiments, ttis analysis can only be applied when Axu remains the same for wild-type and mutant proteins.
ttese methods have allowed us to establish that mechanical unfolding of titin 127 is a three-state process in which the transition state is very similar to the native state (Sect. 8.4.4).
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