|Title||Engineered Bi-Histidine Metal Chelation Sites Map the Structure of the Mechanical Unfolding Transition State of an Elastomeric Protein Domain GB1|
|Publication Type||Journal Article|
|Year of Publication||2012|
|Authors||Shen, T, Cao, Y, Zhuang, S, Li, H|
|Date Published||AUG 22|
Determining the structure of the transition state is critical for elucidating the mechanism behind how proteins fold and unfold. Due to its high free energy, however, the transition state generally cannot be trapped and studied directly using traditional structural biology methods. Thus, characterizing the structure of the transition state that occurs as proteins fold and unfold remains a major challenge. Here, we report a novel (to our knowledge) method that uses engineered bi-histidine (bi-His) metal-binding sites to directly map the structure of the mechanical unfolding transition state of proteins. This method is adapted from the traditional psi-value analysis, which uses engineered bi-His metal chelation sites to probe chemical (un)folding transition-state structure. The phi(M2+) (upsilon)-value is defined as Delta Delta G double dagger(-N)/Delta Delta G(U-N), which is the energetic effects of metal chelation by the bi-His site on the unfolding energy barrier (Delta G double dagger(-N)) relative to its thermodynamic stability (Delta G(U-N)) and can be used to obtain information about the transition state in the mutational site. As a proof of principle, we used the small protein GB1 as a model system and set out to map its mechanical unfolding transition-state structure. Using single-molecule atomic force microscopy and spectrofluorimetry, we directly quantified the effect of divalent metal ion binding on the mechanical unfolding free energy and thermodynamic stability of GB1, which allowed us to quantify phi(M2+)(upsilon)-values for different sites in GB1. Our results enabled us to map the structure of the mechanical unfolding transition state of GB1. Within GB1's mechanical unfolding transition state, the interface between force-bearing beta-strands 1 and 4 is largely disrupted, and the first beta-hairpin is partially disordered while the second beta-hairpin and the alpha-helix remain structured. Our results demonstrate the unique application of psi-value analysis in elucidating the structure of the transition state that occurs during the mechanical unfolding process, offering a potentially powerful new method for investigating the design of novel elastomeric proteins.