1. Department of Physiology and Biophysics, Mayo Foundation, Rochester, Minnesota 55905, USA
2. Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA

Correspondence to: Julio M. Fernandez¹ Correspondence and requests for materials should be addressed to J.M.F. (e-mail: Email: fernandez-julio@mayo.edu).

Abstract

The modular protein titin, which is responsible for the passive elasticity of muscle, is subjected to stretching forces. Previous work on the experimental elongation of single titin molecules has suggested that force causes consecutive unfolding of each domain in an all-or-none fashion^{1, 2, 3, 4, 5, 6}. To avoid problems associated with the heterogeneity of the modular, naturally occurring titin, we engineered single proteins to have multiple copies of single immunoglobulin domains of human cardiac titin⁷. Here we report the elongation of these molecules using the atomic force microscope. We find an abrupt extension of each domain by 7 Å before the first unfolding event. This fast initial extension before a full unfolding event produces a reversible 'unfolding intermediate'. Steered molecular dynamics^{8, 9} simulations show that the rupture of a pair of hydrogen bonds near the amino terminus of the protein domain causes an extension of about 6 Å, which is in good agreement with our observations. Disruption of these hydrogen bonds by site-directed mutagenesis eliminates the unfolding intermediate. The unfolding intermediate extends titin domains by 15% of their slack length, and is therefore likely to be an important previously unrecognized component of titin elasticity.