Flexibility and extensibility in the titin molecule: analysis of electron microscope data.

Muscle elasticity derives directly from titin extensibility, which stems from three distinct types of spring-like behaviour of the I-band portion of the molecule. With progressively greater forces and sarcomere lengths, the molecule straightens and then unfolds, first in the PEVK-region and then in individual immunoglobulin domains. Here, we report quantitative analysis of flexibility and extensibility in isolated titin molecules visualized by electron microscopy. Conformations displayed by molecules dried on a substrate vary from a random coil to rod-like, demonstrating highly flexible and easily deformable tertiary structure. The particular conformation observed depends on the "wettability" of the substrate during specimen preparation: higher wettability favours coiled conformations, whereas lower wettability results in more extended molecules. Extension is shown to occur during liquid dewetting. Statistical methods of conformational analysis applied to a population of coiled molecules gave an average persistence length 13.5(+/-4.5) nm. The close correspondence of this value to an earlier one from light-scattering studies confirms that conformations observed by microscopy closely reflected the equilibrium conformation in solution. Analysis of hydrodynamic forces exerted during dewetting also indicates that the force causing straightening of the molecules and extension of the PEVK-region is in the picoNewton range, whereas unfolding of the immunoglobulin and fibronectin domains may require forces about tenfold higher. The microscope data directly illustrate the relationship between titin conformation and the magnitude of applied force. They also suggest the presence of torsional stiffness in the molecule, which may affect considerations of elasticity.