Mechanochemical coupling cycles underlie the work-generation mechanisms of biological systems and are realized by highly regulated conformational changes of the protein machineries. However, it has been challenging to utilize protein conformational changes to do mechanical work at the macroscopic level in biomaterials, and it remains elusive to construct macroscopic mechanochemical devices based on molecular-level mechanochemical coupling systems. Here, the authors demonstrate that protein folding can be utilized to realize protein’s mechanochemical cycles at both single-molecule and macroscopic levels. Our results demonstrate, for the first time, the successful harnessing of mechanical work generated by protein folding in a macroscopic protein hydrogel device, and the work generated by protein folding compares favorably with the energy output of molecular motors. Our work bridges a gap between single-molecule and macroscopic levels, and paves the way to utilizing proteins as building blocks to design protein-based artificial muscles and soft actuators.

Optical tweezers experiments reveal that the folding of the C-terminal fragment of Top7 (cFr) is context-dependent. Depending on its neighboring sequence, cFr shows very different folding pathways and folding kinetics.

The efficient translocation of the bacterial toxin adenylate cyclase toxin (CyaA) from the bacterial cytosol to the extracellular environment by the type 1 secretion system (T1SS) is essential for the toxin to function. To understand the molecular features that are responsible for the efficient translocation of CyaA, here we used optical tweezers to investigate the mechanical properties and conformational dynamics of the RTX domain of CyaA at the single molecule level. Our results revealed that apo-RTX behaves like an ideal random coil. This property allows the T1SS to translocate RTX without overcoming the enthalpic resistance. In contrast, the folded holo-RTX is mechancially stable, and its folding occurs in a vectorial, cotranslocational fashion starting from its C-terminus. Moreover, our results showed that the folding of holo-RTX generates a stretching force, which can further facilitate the translocation of RTX. Our results highlight the important role played by the Ca2+-triggered folding of RTX in the translocation of RTX and provide mechanistic insights into the mechanical design that governs the efficient translocation of RTX.

Knotted and slipknotted proteins are topologically complex. Understanding their folding and unfolding mechanism has attracted considerable interest. Here we combined protein engineering, single-molecule optical tweezers, and steered molecular dynamics (SMD) simulations to investigate the mechanical unfolding and folding of a slipknotted protein pyruvoyl-dependent arginine decarboxylase (PADC). In its slipknotted structure, PADC contains a long threaded loop (85 residues), which is almost twice the size of the knotting loop. When stretched from its N- and C-termini, the majority of PADC can be readily unfolded in a two-state manner, and the slipknotted structure was untied. A small percentage of PADC unfolded following a three-state pathway involving the formation of an unfolding intermediate state. These unfolding intermediate states showed a broad distribution of contour length increments, suggesting that they did not have a well-defined specific structure. SMD simulations revealed the main free energy barrier to the unfolding of PADC and suggested that the unfolding intermediate states may originate from the frication of polypeptide chain sliding during the process of pulling the threaded loop out of the knotting loop. Upon relaxation, a small percentage of the unfolded and untied PADC polypeptide chain can refold back to its native slipknotted conformation, but a large fraction can only reach a misfolded state. Our results revealed the complexity of the mechanical unfolding and refolding of a slipknotted protein with a long threaded loop.

Mechanical stability of Ca2+-responsive β-roll peptides (RTX) is largely responsible for the Ca2+-dependent mechanical properties of the RTX-based hydrogels.