Gram-positive bacteria can resist large mechanical perturbations during their invasion and colonization by secreting various surface proteins with intramolecular isopeptide or ester bonds. Compared to isopeptide bonds, ester bonds are prone to hydrolysis. It remains elusive whether ester bonds can completely block mechanical extension similarly to isopeptide bonds, or whether ester bonds dissipate mechanical energy by bond rupture. Here, we show that an ester-bond containing stalk domain of Cpe0147 is inextensible even at forces > 2 nN. The ester bond locks the structure to a partially unfolded conformation, in which the ester bond remains largely water inaccessible. This allows the ester bond to withstand considerable mechanical forces and in turn prevent complete protein unfolding. However, the protecting effect might be reduced at non-physiological basic pHs or low calcium concentrations due to destabilizing the protein structures. Inspired by this design principle, we engineer a disulfide mutant resistant to mechanical unfolding under reducing conditions.

Gram-positive bacteria experience considerable mechanical perturbation when adhering to host surfaces during colonization and infection. They have evolved various adhesion proteins that are mechanically robust to ensure strong surface adhesion. Recently, it was discovered that these adhesion proteins contain rare, extra intramolecular covalent bonds that stabilize protein structures and participate in surface bonding. These intramolecular covalent bonds include isopeptides, thioesters, and ester bonds, which often form spontaneously without the need for additional enzymes. With the development of singlemolecule force spectroscopy techniques, the detailed mechanical roles of these intramolecular covalent bonds have been revealed. In this review, we summarize the recent advances in this area of research, focusing on the link between the mechanical stability and function of these covalent bonds in Gram-positive bacterial surface proteins. We also highlight the potential impact of these discoveries on the development of novel antibiotics and chemical biology tools.

Hydrogels are soft materials constructed of physically or chemically crosslinked polymeric net-works with abundant water. The crosslinkers, as the mechanophores that bear and respond to mechanical forces, play a critical role in determining the mechanical properties of hydrogels. Here, we use a polyprotein as the crosslinker and mechanophore to form covalent polymer hydrogels in which the toughness and fatigue fracture are controlled by the mechanical unfolding of polyproteins. The protein Parvimonas sp. (ParV) is super stable and remains folded even at forces > 2 nN; however, it can unfold under loading forces of ~100 pN at basic pH values or low calcium concentrations due to destabilization of the protein structures. Through tuning the protein unfolding by pH and calcium concentrations, the hydrogel exhibits differences in modulus, strength, and anti-fatigue fracture. We found that due to the partially unfolding of ParV, the Young’s modulus decreased at pH 9.0 or in the presence of EDTA (Ethylene Diamine Tetraacetic Acid), moreover, because partially unfolded ParV can be further completely unfolded due to the mechanically rupture of ester bond, leading to the observed hysteresis of the stretching and relaxation traces of the hydrogels, which is in line with single-molecule force spectroscopy experiments. These results display a new avenue for designing pH- or calcium-responsive hydrogels based on proteins and demonstrate the relationship between the mechanical properties of single molecules and macroscopic hydrogel networks.