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A biological system is essentially in non-equilibrium. Since microscopic internal variables of the system emerges in a non-equilibrium process in addition to macroscopic ones, experimental approaches in single-molecule scale is more suited for probing such a process. In the present study, transformation of a single modular protein molecule induced by an external force is studied using viscoelasticity measurement technique based on atomic force microscopy (AFM) with magnetic modulation of the cantilever. A titin (or connectin) single molecule in muscle sarcomere is a well-known modular protein that consists of immunoglobulin (Ig) and fibronectin-3 (Fn3) domains. Upon stretching with an external tensile load, the Ig domains undergo a transition to an unfolding intermediate state in which only the hydrogen bonds bridging A and B strands are broken. It was reported that restoration of the structure to a native form upon unloading typically requires time of the order of 10 ms. In the present study, the transformation to the intermediate was hardly reflected in the measured elasticity profile when the modulation amplitude of the AFM cantilever was small enough. Consequently the elasticity data integrated with the elongation distance exhibited a discrepancy with the DC force profile. When the amplitude was increased the elasticity profile exhibited a characteristic peaking upon the transition, which is consistent with the reports by other groups. It is a mystery why the transition was detected with the modulation much faster than the above-mentioned refolding time-scale under a bias of strong tensile load that is to lower the refolding rate by roughly three orders of magnitude from the unloaded value. The effect of modulation amplitude on the folding dynamics will be discussed. Upon further loading usually the domains transform to a fully unraveled state instantaneously. However, only one domain was observed to anomalously relax taking longer than 100 ms. Its detailed process will be discussed based on the theory of dynamics of a single polymer chain. |