Pulling single molecules of titin by AFM--recent advances and physiological implications.

Perturbation of a protein away from its native state by mechanical stress is a physiological process immanent to many cells. The mechanical stability and conformational diversity of proteins under force therefore are important parameters in nature. Molecular-level investigations of "mechanical proteins" have enjoyed major breakthroughs over the last decade, a development to which atomic force microscopy (AFM) force spectroscopy has been instrumental. The giant muscle protein titin continues to be a paradigm model in this field. In this paper, we review how single-molecule mechanical measurements of titin using AFM have served to elucidate key aspects of protein unfolding-refolding and mechanisms by which biomolecular elasticity is attained. We outline recent work combining protein engineering and AFM force spectroscopy to establish the mechanical behavior of titin domains using molecular "fingerprinting." Furthermore, we summarize AFM force-extension data demonstrating different mechanical stabilities of distinct molecular-spring elements in titin, compare AFM force-extension to novel force-ramp/force-clamp studies, and elaborate on exciting new results showing that AFM force clamp captures the unfolding and refolding trajectory of single mechanical proteins. Along the way, we discuss the physiological implications of the findings, not least with respect to muscle mechanics. These studies help us understand how proteins respond to forces in cells and how mechanosensing and mechanosignaling events may proceed in vivo.