Mechanical unfolding of TNfn3: The unfolding pathway of a fnIII domain probed by protein engineering, AFM and MD simulation

Protein engineering (D-value analysis combined with single molecule atomic force microscopy (AFM) was used to probe the molecular basis for the mechanical stability of TNfn3, the third fibronectin type III domain from human tenascin. This approach has been adopted previously to solve the forced unfolding pathway of a titin immunoglobulin domain, TI 127. TNfn3 and TI 127 are members of different protein superfamilies and have no sequence identity but they have the same P-sandwich structure consisting of two antiparallel beta-sheets. TNfn3, however, unfolds at significantly lower forces than TI 127. We compare the response of these proteins to mechanical force. Mutational analysis shows that, as is the case with TI 127, TNfn3 unfolds via a force-stabilised ihtermediate. The key event in forced unfolding in TI 127 is largely the breaking of hydrogen bonds and hydrophobic interactions between the A' and G-strands. The mechanical (D-value analysis and molecular dynamics simulations reported here reveal that significantly more of the TNfn3 molecule contributes to its resistance to force. Both AFM experimental data and molecular dynamics simulations suggest that the rate-limiting step of TNfn3 forced unfolding reflects a transition from the extended early intermediate to an aligned intermediate state. As well as losses of interactions of the A and G-strands and associated loops there are rearrangements throughout the core. As was the case for TI 127, the forced unfolding pathway of TNfn3 is different from that observed in denaturant studies in the absence of force. (c) 2005 Elsevier Ltd. All rights reserved.