Modeling AFM-Induced PEVK Extension and the Reversible Unfolding of Ig/FNIII Domains in Single and Multiple Titin Molecules

Abstract

Molecular elasticity is associated with a select number of polypeptides and proteins, such as titin, Lustrin A, silk fibroin, and spider silk dragline protein. In the case of titin, the globular (Ig) and non-globular (PEVK) regions act as extensible springs under stretch; however, their unfolding behavior and force extension characteristics are different. Using our time-dependent macroscopic method for simulating AFM-induced titin Ig domain unfolding and refolding, we simulate the extension and relaxation of hypothetical titin chains containing Ig domains and a PEVK region. Two different models are explored: 1) a series-linked WLC expression that treats the PEVK region as a distinct entropic spring, and 2) a summation of N single WLC expressions that simulates the extension and release of a discrete number of parallel titin chains containing constant or variable amounts of PEVK. In addition to these simulations, we also modeled the extension of a hypothetical PEVK domain using a linear Hooke's spring model to account for "enthalpic" contributions to PEVK elasticity. We find that the modified WLC simulations feature chain length compensation, Ig domain unfolding/refolding, and force-extension behavior that more closely approximate AFM, laser tweezer, and immunolocalization experimental data. In addition, our simulations reveal the following: 1) PEVK extension overlaps with the onset of Ig domain unfolding, and 2) variations in PEVK content within a titin chain ensemble lead to elastic diversity within that ensemble.

An energetic model for macromolecules unfolding in stretching experiments

“…Figure 2 shows (in a log scale, stressing the differences at low values of the force) that, while for low forces (F , 10 21 pN) the introduced approximation is significant (when compared with the approximation in [49]), for larger forces the approximation is of the same order (actually approximating better than [49] the WLC expression) as the one proposed by [49]. Because in the low force regime the elasticity is mainly regulated by the PEVK and tertiary structure elasticity (see [50,22] for details), this approximation appears inessential in both the qualitative and the quantitative analysis of the behaviour during the large-force unfolding regime of interest in this paper. Moreover, we remark that the approximation (3.1) has been shown to be inefficient in the low force regime in [51], where the authors introduce a Mooney-Rivlin-type correction to the WLC constitutive law.…”

Pulling single molecules of titin by AFM—recent advances and physiological implications

“…In this example, the persistence length for the PEVK region was 1.0 nm, and the contour length was 80 nm. One conclusion drawn from these experiments is that the unique sequences in titin generate elastic forces mainly according to an entropic-spring mechanism, although evidence abounds suggesting that additional factors determine the elasticity of the PEVK domain [27, 58,72,76,92,132,137]. In any case, these and related studies [27, 58,62,66,67,92,135] clearly demonstrated that the PEVK domain and the N2B us extend at forces at which the Ig domain unfolding probability is still very low.…”

“…An invaluable contribution to understanding the critical events leading to mechanical unfolding has come from steered molecular dynamics (SMD) simulations and other modeling approaches [4,28,31,32,54,80,81,85,103,104,137], which have used the available atomic structures of the titin Ig domains I1 [88] and I91 [45]. For I91, SMD simulations showed that mechanical force application first breaks two backbone hydrogen bonds between the beta-strands A and B-an event that is sometimes projected in AFM recordings as a small "hump" during the force rise (see Figs.…”

Myomesin is a Molecular Spring with Adaptable Elasticity

“…There is a tendency for the domains unfolded first to have slightly lower unfolding forces than the subsequent domains, which can be predicted from theoretical studies. 39 An average increase in force of 7.4 pN (Figure 4(c)) was determined from one peak to the next, which may result from the decrease in unfolding probability, as more domains are The (My10) 9 molecules appear longer and slightly less compact than the (My6) 8 ones, which can be explained by the higher number of modules and by the longer linker length in the (My10) 9 polyprotein (35 aa) compared to (My6) 8 (15 aa). In contrast, rotary metal-shadowed images of the (My6-EH) 4 construct reveal the My6 modules as four small globules (arrowheads), evidently connected by an "invisible thread" (i.e.…”