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 substratesin vivois 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 degrees C. Our data providein vivoevidence 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.