Reduced to the Single Molecule Level

tte sarcomere is the basic contractile unit of muscle. Early studies showed that this relatively simple contraction/extension machine (i.e., it works in a single dimension) is also highly elastic (Huxley and Hanson 1954). tte pioneering work by Wanget al. (1979) and Maruyama etal. (1981) demonstrated that the so-called ┬źpassive elasticity┬╗ of muscle (i.e., generation of restoring forces that resist stretch independently of ATP) is mainly mediated by titin, a giant protein (more than 3 MDa, the longest polypeptide known to date) that spans half a sarcomere (approximately 1 ^m; from the Z disk to the M line) and acts as a molecular spring (Fig. 8.2c). Passive elasticity plays an important role in muscle function since, typically, a muscle actively contracts against the elastic strain of a passively elongating muscle, ttis property ensures that the sarcomere recovers its initial dimensions on muscle relaxation (Tskhovrebova and Trinick 2003).

One of the remarkable feats of SMFS has been the reconstruction of the passive elasticity of intact myofibrils on the basis of a simple scaling up from the mechanical properties of single molecules of titin (Li et al. 2002). tte mechanics of single titin molecules was reconstituted in turn from the mechanical properties of representative elements of its elastic region (i.e., N2, PEVK, and the proximal and distal Ig regions relative to the N-terminus of the protein), showing that titin behaves very differently from a Hookean spring (e.g., the cantilever of an AFM; Fig. 8.6a, panel 1). It is essentially an entropic spring in which force and extension do not follow a linear relationship.

trough this reductionist approach it was possible to explain muscle passive elasticity, a macroscopic property, from the additive mechanical properties of a single sarcomeric protein at the single-molecule level. According to this model, within the physiological range of sarcomere extension (i.e., low forces below 4 pN) unfolding would rarely happen; thus most of the elasticity of titin in the physiological range would result from the entropic elasticity of straightening the Ig domains in the Iband, and of extending its unique sequences (PEVK and N2B). Mechanical unfolding of the Ig modules would basically function as a buffer of both length and additional entropic elasticity. Ms "shock-absorber" effect would only occur at a high force, i.e., in nonphysiological conditions, to prevent damage of the sarcomere (i.e., it is essentially a safety mechanism), tterefore, in response to axial tension, titin behaves as a multistage spring that adjusts both its length and apparent stiffness by virtue of its particular modular design.

tte example of titin elasticity shows how single-molecule experiments can be used to elucidate, at a more fundamental level, the physiological function of a protein through the biophysical dissection of a complex hierarchical system.

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