We next investigated the unfolding process starting from the native MccJ25 structure (PDB ID 1Q71) with the covalent isopeptide bond between residues Gly1 and Glu8 hydrolyzed to examine how readily it transitions to pre-tadpole and unfolded structures (Figure ). For eight replicas, the pre-lasso configuration remained stable for ~150-500 ns, which is notably longer than the previously reported unfolding in less than 4 ns. Within this shallow metastable basin, the distance between residues Gly1 and Glu8 fluctuates apart and returns to the prelasso state while the β-hairpin remains stable. During this time, approximately half of the replicas included the formation of transient pre-tadpoles but returned to pre-lassos. After ~500 ns, the β-hairpin is lost, and the system does not return to a pre-lasso form in 7.2 µs (Figure ), which is also demonstrated in the secondary structure map (Figure ). Thus, a metastable minimum containing pre-lasso structures does exist, but it represents less than 1% of the above sampled conformations (Table ), suggesting it is rare relative to the entropically-favored unfolded phase space. starting prelasso (t = 0 ns); fluctuating between pre-lasso and pre-tadpole (t < 500 ns); completely unfolded (t > 500 ns).