Titin, a giant filamentous polypeptide, is believed to play a fundamental role in maintaining sarcomeric structural integrity and developing what is known as passive force in muscle. Measurements of the force required to stretch a single molecule revealed that titin behaves as a highly nonlinear entropic spring. The molecule unfolds in a high-force transition beginning at 20 to 30 piconewtons and refolds in a low-force transition at approximately 2.5 piconewtons. A fraction of the molecule (5 to 40 percent) remains permanently unfolded, behaving as a wormlike chain with a persistence length (a measure of the chain's bending rigidity) of 20 angstroms. Force hysteresis arises from a difference between the unfolding and refolding kinetics of the molecule relative to the stretch and release rates in the experiments, respectively. Scaling the molecular data up to sarcomeric dimensions reproduced many features of the passive force versus extension curve of muscle fibers.

The assembly mechanisms of amyloid fibrils, tissue deposits in a variety of degenerative diseases, is poorly understood. With a simply modified application of the atomic force microscope, we monitored the growth, on mica surface, of individual fibrils of the amyloid ␤25-35 peptide with near-subunit spatial and subsecond temporal resolution. Fibril assembly was polarized and discontinuous. Bursts of rapid (up to 300-nm ؊1 ) growth phases that extended the fibril by Ϸ7 nm or its integer multiples were interrupted with pauses. Stepwise dynamics were also observed for amyloid ␤1-42 fibrils growing on graphite, suggesting that the discontinuous assembly mechanisms may be a general feature of epitaxial amyloid growth. Amyloid assembly may thus involve fluctuation between a fast-growing and a blocked state in which the fibril is kinetically trapped because of intrinsic structural features. The used scanning-force kymography method may be adapted to analyze the assembly dynamics of a wide range of linear biopolymers.atomic force microscopy ͉ beta-amyloid ͉ growth dynamics ͉ self-assembly

Titin is a giant elastic protein that is responsible for the majority of passive force generated by the myocardium. Titin's force is derived from its extensible I-band region, which, in the cardiac isoform, comprises three main extensible elements: tandem Ig segments, the PEVK domain, and the N2B unique sequence (N2B-Us). Using atomic force microscopy, we characterized the single molecule force-extension curves of the PEVK and N2B-Us spring elements, which together are responsible for physiological levels of passive force in moderately to highly stretched myocardium. Stretch-release force-extension curves of both the PEVK domain and N2B-Us displayed little hysteresis: the stretch and release data nearly overlapped. The force-extension curves closely followed worm-like chain behavior. Histograms of persistence length (measure of chain bending rigidity) indicated that the single molecule persistence lengths are ϳ1.4 and ϳ0.65 nm for the PEVK domain and N2B-Us, respectively. Using these mechanical characteristics and those determined earlier for the tandem Ig segment (assuming folded Ig domains), we modeled the cardiac titin extensible region in the sarcomere and calculated the extension of the various spring elements and the forces generated by titin, both as a function of sarcomere length. In the physiological sarcomere length range, predicted values and those obtained experimentally were indistinguishable.Titin forms a striated muscle-specific myofilament that develops passive force in response to sarcomere stretch (for a recent review with original citations, see Ref. 1). Titin's force is generated by serially linked and mechanically distinct spring elements (2). Tandem Ig segments (tandemly arranged Ig-like domains) and the PEVK domain (rich in proline, glutamate, valine, and lysine residues) are spring elements found in both cardiac and skeletal muscle titins (3, 4) that vary in length in different isoforms of titin. For example, in human, the PEVK domain varies from 188 residues in the cardiac-specific N2B isoform to 2181 residues in skeletal soleus muscle (2). The cardiac-specific N2B unique sequence (N2B-Us) 1 forms a third spring element in cardiac titins and provides extensibility at the upper range of physiological sarcomere lengths in the heart (5-7). The three-spring system of cardiac titin results in a unique force-extension curve that underlies the majority of the physiological passive tensions of the myocardium, and the variable-length tandem Ig and PEVK elements allows passive tension to be adjusted so that it matches the mechanical demands placed on normal and diseased myocardium (8, 9).Immunoelectron microscopy has shown that in slack sarcomeres (no external force), the tandem Ig segments are in a "contracted" state. When the sarcomeres are stretched, the segments greatly extend (4) due to unbending of linkers between folded Ig domains (4) and possibly to limited domain unfolding (10). The tandem Ig segments exhibit worm-like chain (WLC) behavior with a persistence length (measure of chain bendi...