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Abstract

Protein folding is a fundamental biological self-assembly process in which a disordered polypeptide chain adopts a defined three-dimensional structure to perform its biological function. Over the past two decades, mechanical force has been widely used as a denaturant to investigate protein stability and folding mechanisms. Single-molecule force spectroscopy (SMFS) has become an important tool for probing the forces and conformational dynamics of biomolecules. By applying force, the protein energy landscape is perturbed, leading to conformational transitions, which can be directly measured using SMFS at the single-molecule level, avoiding ensemble averaging in bulk experiments. Among SMFS techniques, optical tweezers provide high spatial and force resolution, allowing detailed analysis of protein unfolding and refolding pathways. This method also enables the investigation of individual domains within multidomain proteins, such as adenylate cyclase toxin, a Ca²⁺-binding member of the repeats-in-toxin (RTX) family composed of five RTX blocks. In this study, optical tweezers combined with protein engineering were used to characterize the mechanical properties of RTX block II within the truncated construct RTX–II–III1333–4βcap. This construct offered a simplified and experimentally tractable system, while preserving key structural features of the native protein. Results revealed multiple unfolding and refolding pathways with distinct contour length changes, exhibiting both two-state and three-state behavior. Notably, RTX block II in the truncated construct predominantly followed a three-state unfolding pathway, in contrast to its behavior in the wild-type RTX–II–V construct. The presence of a folding intermediate in the three-state pathway significantly reduced the refolding rate.