Stability of tandem Ig domain unfolding within proximal Ig segment of titin.

Abstract

Titin is a giant filamentous protein contributing to the passive elasticity of muscle and is composed of approximately 300 serially linked immunoglobulin-like domains. The tandem domains of titin's proximal immunoglobulin (Ig) segment have been shown to separate at relatively low forces and thus contribute to the protein's elasticity. While titin's elastic behavior is attributed to extension of the proximal Ig segment and PEVK region beyond passive slack length, the potential unfolding of individual Ig domains has been posited to contribute to even greater extensibility. Computational modeling and mechanical stability testing have demonstrated Ig domain unfolding under physiologically relevant force, with neighboring domains exhibiting variable degrees of stability. Thermal and chemical stability experiments were conducted on domains I65-I70 of the proximal tandem Ig segment to evaluate the stability of adjacent domains. Testing revealed variable free energies of unfolding, with I65 demonstrating the lowest thermal and chemical stability out of all domains of interest, and I67 demonstrating the highest. The quantification of each domain's relative stability helps elucidate the role of the proximal Ig segment in titin's elasticity and can be used to assess the unfolding potential of other domains along the protein's extensible regions.