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 ∼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 role of hydrophobic and polar interactions in providing thermodynamic stability to folded proteins has been intensively studied, but the relative contribution of these interactions to the mechanical stability is less explored. We used steered molecular dynamics simulations with constant-velocity pulling to generate force-extension curves of selected protein domains and monitor hydrophobic surface unravelling upon extension. Hydrophobic contribution was found to vary between one fifth and one third of the total force while the rest of the contribution is attributed primarily to hydrogen bonds. Moreover, hydrophobic force peaks were shifted towards larger protein extensions with respect to the force peaks attributed to hydrogen bonds. The higher importance of hydrogen bonds compared to hydrophobic interactions in providing mechanical resistance is in contrast with the relative importance of the hydrophobic interactions in providing thermodynamic stability of proteins. The different contributions of these interactions to the mechanical stability are explained by the steeper free energy dependence of hydrogen bonds compared to hydrophobic interactions on the relative positions of interacting atoms. Comparative analyses for several protein domains revealed that the variation of hydrophobic forces is modest, while the contribution of hydrogen bonds to the force peaks becomes increasingly important for mechanically resistant protein domains.

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 ∼1.4 and ∼0.65 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.

Easily accessible green-light activatable (>500 nm) photocages based on red-shifted, π-extended coumarin scaffolds are developed with uncaging efficiencies similar to those of recently introduced BODIPY derivatives. The photocages possess increased aqueous solubility, high absorption coefficients within the 450–600 nm range, and exceptionally high two-photon cross sections.

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 β25–35 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 ≈7 nm or its integer multiples were interrupted with pauses. Stepwise dynamics were also observed for amyloid β1–42 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.