Abstract
The force-induced unfolding of calmodulin (CaM) was investigated at atomistic details with steered molecular dynamics. The two isolated CaM domains as well as the full-length CaM were simulated in N-C-terminal pulling scheme, and the isolated N-lobe of CaM was studied specially in two other pulling schemes to test the effect of pulling direction and compare with relevant experiments. Both Ca2+-loaded CaM and Ca2+-free CaM were considered in order to define the Ca2+ influence to the CaM unfolding. The results reveal that the Ca2+ significantly affects the stability and unfolding behaviors of both the isolated CaM domains and the full-length CaM. In Ca2+-loaded CaM, N-terminal domain unfolds in priori to the C-terminal domain. But in Ca2+-free CaM, the unfolding order changes, and C-terminal domain unfolds first. The force-extension curves of CaM unfolding indicate that the major unfolding barrier comes from conquering the interaction of two EF-hand motifs in both N- and C- terminal domains. Our results provide the atomistic-level insights in the force-induced CaM unfolding and explain the observation in recent AFM experiments.
Highlights
The force-induced Ca2+-loaded CaM unfolding in its isolated and full-length state has been investigated experimentally with the single molecule atomic force microscope (AFM) techniques [23,28] It is shown that there is a major energy barrier in the force-induced unfolding of each isolated CaM domain and there exist two distinct force peaks in the extension-force curves of the full-length CaM which correspond to the unfolding of two globular CaM domains which can be characterized by the difference extension length fitted with the wormlike chain model
Some fine-tuned force peaks, which are responsible for the helix unfolding and reorientation of the globular domain, are found in the simulation results which were not visible in AFM studies due to the spatial and force resolution of the experiments
The maximum forces to unfold the CaM constructs in our simulations are about 10 times larger than those observed in the AFM experiments
Summary
Our simulation results suggest that the unfolding order of N- and C-terminal domains are related with the Ca2+ binding states, and the interactions between the two EF-hand motifs compose the main energy barrier on CaM unfolding. Monitoring the conformational change of CaM unfolding, it is found that when the first main peak force FHolo-max1 occurs, the backbone H-bonds between Ile27 and Ile63 in N-lobe began to vanish (Fig. 3A-IV), and the contact area of EF1 and EF2 droped to zero rapidly (Fig. 3A-II), indicating that the two coupled EF-hand motifs were dissociating.
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