Role of native topology investigated by multiple unfolding simulations of four SH3 domains

The relative importance of amino acid sequence and native topology in the u nfolding process of two SH3 domains and two circular permutants was investi gated by 120 molecular dynamics runs at 375 K for a total simulation time o f 0.72 mus. The alpha -spectrin (aSH3) and src SH3 (sSH3) domains, which ha ve the same topology and a sequence identity of only 34%, show similar unfo lding pathways. The disappearance of the three-stranded antiparallel beta - sheet is the last unfolding event in agreement with a large repertoire of k inetic data derived from point mutations as well as glycine insertions and disulfide crosslinks. Two alternative routes of beta -sheet unfolding have emerged from the analysis of the trajectories. One is statistically preferr ed in aSH3 (n-src loop breaks before distal hairpin) and the inverse in sSH 3. An elongation of the beta2-beta3 hairpin was observed during the unfoldi ng of sSH3 at 375 K and in 300 K simulations started from the putative tran sition state of sSH3 in accord with unusual kinetic data for point mutation s at the n-src loop. The change of connectivity in the permutants influence d the sequence of unfolding events mainly at the permutation site. Regions where the connectivity remained unaffected showed the same chronology of co ntact disappearance. Taken together with previous folding simulations of tw o designed three-stranded antiparallel beta -sheet peptides, these results indicate that, at least for small beta -sheet proteins, the folding mechani sm is primarily defined by the native state topology, whilst specific inter actions determine the statistically predominant folding route. (C) 2001 Aca demic Press.