The mechanical stability of immunoglobulin and fibronectin III domains in the muscle protein titin measured by atomic force microscopy

The domains of the giant muscle protein titin (connectin) provide interacti on sites for other sarcomeric proteins and fulfill mechanical functions. In this paper we compare the unfolding forces of defined regions of different titin isoforms by single-molecule force spectroscopy. Constructs comprisin g six to eight immunoglobulin (Ig) domains located in the mechanically acti ve I-band part of titin are compared to those containing fibronectin III (F n3) and lg domains from the A-band part. The high spatial resolution of the atomic force microscope allows us to detect differences in length as low a s a few amino acids. Thus constructs of different lengths may be used as mo lecular rulers for structural comparisons with other modular proteins. The unfolding forces range between 150 and 300 pN and differ systematically bet ween the constructs. Fn3 domains in titin exhibit 20% lower unfolding force s than Ig domains. Fn3 domains from tenascin, however, unfold at forces onl y half those of titin Fn3 domains. This indicates that the tightly folded t itin domains are designed to maintain their structural integrity, even unde r the influence of stretching forces. Hence, at physiological forces, unfol ding is unlikely unless the forces are applied for a long time (longer than minutes).