Single Molecule Study On The Mechanical Response Of The Sixth Domain Of Adseverin

Adseverin,also known as scinderin,was first isolated from chromaffin cells of bovine adrenal in 1986.Adseverin is an actin-binding protein,which can sever,cap and uncap actin filaments,thus regulating the architecture of the actin cytoskeleton.Adseverin can affect cellular processes,such as cytoskeleton rearrangement,exocytosis and cell differentiation by regulating actin organisation.Adseverin needs to constantly switch between activated and inactivated conformations,which are dependent on calcium ion concentration([Ca²⁺])to function in cellular processes.In the absence of Ca²⁺,adseverin is in globular form and could not bind with actin.Upon calcium binding,adseverin will exhibit a pearl-necklace-like form and interact with actin.Force will be generated and transferred inside adseverin,as well as between adseverin and actin when adseverin performs its functions.However,adseverin will not bind with actin in the absence of Ca²⁺because of the steric clashes between sixth domain of adseverin?A6?and actin.Consequently,the steric clashes should be broken to facilitate the interaction of adseverin with actin.Compared with its analogue,gelsolin,the sixth domain of adseverin lacks an?-helix in the C terminus.This characteristic results in the distinct differences of severing rates and severing temperature sensitivity between the two proteins.Hence,it is very important to study mechanical stability of A6.Single-molecule force spectroscopy?SMFS?techniques,such as those based on atomic force microscopy?AFM?and magnetic tweezers?MT?,have become very important tools to study the mechanical properties of individual molecules at the single-molecule level.SMFS techniques have been successfully applied to investigate the nature of protein unfolding/folding,DNA melting and nucleic acid-protein interactions.These information have deepened the understanding on the mechanism of several important biological processes.Here,we studied the mechanical properties of A6 at the single-molecule level using AFM-and MT-based SMFS.The unfolding forces of A6 at different[Ca²⁺]and the unfolding pathway of A6 were obtained.Our findings indicated that the binding of A6 to Ca²⁺was a fast equilibrium process,and Ca²⁺could enhance the mechanical stability of A6.We also found that the unfolding pathway of A6 probed by AFM was different from that by MT.AFM-based SMFS results show that A6 mainly unfolded in a two-state unfolding manner in the absence of Ca²⁺.When the concentration of Ca²⁺high enough(e.g.,the ratio between Ca²⁺and A6 reach 1:1 or above),the percentage of the thee-state unfolding mode increased.While the results obtained by MT-based SMFS show that A6 mainly unfolded in a three-state unfolding manner.And the three-state unfolding of A6 become dominate at a lower external force.The remarkable difference in the unfolding mode probed by AFM and MT may be due to the distinct loading rates of AFM and MT.This result also helps us understand the loading rate dependence of the unfolding process.