HAROLD P. ERICKSON Departmento of Cell Biology, Duke University Medical Center, Durham, NC 27705
The elastic protein titin comprises a tandem array of fibronectin type m and immunoglobulin domains, which are structurally similar 7-strand fl-sandwiches. A proposed mechanism for stretching titin, by sequential denaturation of individual fibronectin type II-immunoglobulin domains in response to applied tension, is analyzed here quantitatively. The folded domain is =4 nm long, and the unraveled polypeptide can extend to 29 nm, providing a 7-fold stretch over the relaxed length. Elastic recoil is achieved by refolding of the denatured domains when the force is released. The critical force required to denature a domain is calculated to be 3.5-5 pN, based on a net free energy for denaturation of 7-14 kcal/mol, plus 5 kcal/mol to extend the polypeptide (1 cal =4.184 J). This force is comparable to the 2- to 7-pN force generated by single myosin or kinesin molecules. The force needed to pull apart a noncovalent protein-protein interface is estimated here to be 10-30 pN, implying that titin will stretch internally before the molecule is pulled from its attachment at the Z band. Many extracellular matrix and cell adhesion molecules, such as fibronectin, contain tandem arrays of fibronectin type III domains. Both single molecules and matrix fibers should have elastic properties similar to titin. The 3000-kDa titin and the related muscle proteins twitchin and projection are composed of tandem repeats of fibronectin type III (FN3) domains, each containing 98-102 amino acids, interspersed with 91-95 amino acid repeats homologous to immunoglobulin (Ig) constant domains (1,2). The complete sequence of twitchin is known (3), and partial sequence data are available for titin (4). A number of extracellular matrix proteins, such as fibronectin, tenascin, and several collagens, and cell adhesion proteins of the immunoglobulin superfamily contain tandem FN3 repeats (5). The titin molecule is thought to function as an elastic element, keeping the myosin filament centered in the sarcomere (6, 7). If the elasticity of titin is based on the structure of the FN3 domain, all of these proteins should have similar elastic properties. Soteriou et al. (8) recently proposed that the elastic stretching of titin must involve reversible unfolding of individual FN3 and Ig domains. No other mechanism seemed possible to achieve the 4-fold extension of the I-band segment observed in stretched muscle fibers. They presented experimental evidence that titin underwent an abrupt denaturation in guanidine hydrochloride corresponding to a free energy of 10 kcal/mol per domain (1 cal = 4.184 J) and compared this energy to that estimated from passive elasticity of muscle. The present analysis, initiated independently, addresses the same basic mechanism and extends the analysis. Additional approaches are developed here to estimate the distance that each domain can be stretched, to calculate the force required to unravel FN3 domains, and to compare this force to forces developed by motor molecules and sustained by protein bonds.