Figure 5.

Cysteine-specific crosslinking of DnaJ and substrate into the substrate-binding cavity of DnaK. DnaK-Q424C, coupled to the heterobifunctional crosslinker BPIA by means of Cys-424, and similarly treated wild-type DnaK (WT) were incubated in the dark at 30°C with combinations of proteins [σ³², 5 μM; trigger factor (TF), 5 μM; DnaJ, 0.5, 1, or 5 μM], peptides (σ³²-H107-N120; σ³²-Q132-Q144, 25 μM), and ATP (5 mM) as indicated by + and − above the lanes. Crosslinking was induced by UV and products were analyzed on SDS/10% polyacrylamide gels (A and B). (Upper) Silver-stained SDS/polyacrylamide gels; (Lower) immunoblots developed with DnaK-, DnaJ-, and σ³²-specific antisera (α-DnaK, etc.). Indicated are positions of molecular weight markers (left) and DnaK, trigger factor (TF), DnaJ, and σ³² (right). Asterisks and open and filled arrowheads denote DnaK-DnaK, DnaK-σ³², and DnaK-DnaJ crosslinks, respectively.