Elasticity and unfolding of single molecules of the giant muscle protein titin

Abstract

The giant muscle protein titin, also called connectin, is responsible for the elasticity of relaxed striated muscle, as well as acting as the molecular scaffold for thick-filament formation. The titin molecule consists largely of tandem domains of the immunoglobulin and fibronectin-III types, together with specialized binding regions and a putative elastic region, the PEVK domain. We have done mechanical experiments on single molecules of titin to determine their visco-elastic properties, using an optical-tweezers technique. On a fast (0.1s) timescale titin is elastic and force-extension data can be fitted with standard random-coil polymer models, showing that there are two main sources of elasticity: one deriving from the entropy of straightening the molecule; the other consistent with extension of the polypeptide chain in the PEVK region. On a slower timescale and above a certain force threshold, the molecule displays stress-relaxation, which occurs in rapid steps of a few piconewtons, corresponding to yielding of internal structures by about 20 nm. This stress-relaxation probably derives from unfolding of immunoglobulin and fibronectin domains.

Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers

“…The polystyrene beads used in the tweezers are much larger (radius of curvature ~500 nm or bigger) than an typical AFM tip (radius of curvature ~10 nm) and therefore more space between the ends of the sample is required to avoid non-specific interactions between the tethering surfaces. Until the approach reported here (and used in Cecconi et al 2005), the only proteins amenable to study with the optical tweezers have been micrometer-long molecules naturally organized in linear arrays of hundreds of globular domains, such as titin (Tskhovrebova et al 1997;Kellermayer et al 2000). These experiments have revealed important aspects of the overall mechanical properties of these molecules, but failed to provide information on specific domain behavior due to heterogeneity of these naturally occurring polymers.…”

Lattice Model Studies of Force-Induced Unfolding of Proteins

“…The initial experiments showed that, upon application of force, the multimodular protein titin unfolds one domain at a time. 1,3 Similar experiments on a number of proteins have verified that unfolding at the single molecule level can be monitored by subjecting the folded proteins to tension. 4,[7][8][9][10] The AFM experiments, which showed the characteristic sawtooth pattern in the force-extension (f -z) curves, were interpreted using the wormlike chain (WLC) model for the domains.…”

“…Hysteresis during Stretch-Release Cycles. The stretching of titin by optical tweezers 2,3 showed that when the unfolded protein is released, the molecule does not spontaneously refold. Upon decreasing the force, its extension initially decreases by about 50% compared to the fully stretched state.…”

“…4,[7][8][9][10] The AFM experiments, which showed the characteristic sawtooth pattern in the force-extension (f -z) curves, were interpreted using the wormlike chain (WLC) model for the domains. [1][2][3] The complete unraveling of the weakest domains was inferred from the spacing between the saw-tooth patterns. Adaptation of Flory estimate of the size of the polypeptide chain can be used to predict the expected spacing assuming that complete unfolding of individual domains occurs as long as sufficient force is applied.…”

“…[1][2][3][4][5][6] These nanomanipulation techniques, in which proteins are pulled using atomic force microscopy (AFM) or optical tweezers, can be used to map the free energy landscape of biomolecules at the single molecule level. To date, these experiments have provided measurements of the forces required to stretch the molecule by a certain distance.…”