Mechanical unfolding intermediates in titin modules

The modular protein titin, which is responsible for the passive elasticity of muscle, is subjected to stretching forces. Previous work on the experime ntal elongation of single titin molecules has suggested that force causes c onsecutive unfolding of each domain in an all-or-none fashion(1-6). To avoi d problems associated with the heterogeneity of the modular, naturally occu rring titin, we engineered single proteins to have multiple copies of singl e immunoglobulin domains of human cardiac titin(7). Here we report the elon gation of these molecules using the atomic force microscope. We find an abr upt extension of each domain by similar to 7 Angstrom before the first unfo lding event. This fast initial extension before a full unfolding event prod uces a reversible 'unfolding intermediate'. Steered molecular dynamics(8,9) simulations show that the rupture of a pair of hydrogen bonds near the ami no terminus of the protein domain causes an extension of about 6 Angstrom, which is in good agreement with our observations. Disruption of these hydro gen bonds by site-directed mutagenesis eliminates the unfolding intermediat e. The unfolding intermediate extends titin domains by similar to 15% of th eir slack length, and is, therefore a likely to be an important previously unrecognized component of titin elasticity.