IbpB‐bound substrate release in living cells as revealed by unnatural amino acid‐mediated photo‐crosslinking

Small heat shock proteins (sHSPs) are known to bind non‐native substrates and prevent irreversible aggregation in an ATP‐independent manner. However, the dynamic interaction between sHSPs and their substrates in vivo is less studied. Here, by utilizing a genetically incorporated crosslinker, we characterized the interaction between sHSP IbpB and its endogenous substrates in living cells. Through photo‐crosslinking analysis of five Bpa variants of IbpB, we found that the substrate binding of IbpB in living cells is reversible upon short‐time exposure at 50 °C. Our data provide in vivo evidence that IbpB engages in dynamic substrate release under nonstress conditions and suggest that photo‐crosslinking may be a suitable method for investigating dynamic interaction between molecular chaperones and their substrates in living cells.