Fig. 4: Isolated JD and CTD proteins are monomeric.
From: Regulatory inter-domain interactions influence Hsp70 recruitment to the DnaJB8 chaperone
a Cartoon schematic for the full-length DnaJB8 and domain fragments JD1–82 and CTD170–232. b Representative SEC profiles of JD1–82 (red), CTD170–232 (green), and LMW standards (blue). JD1–82 and CTD170–232 elute at apparent molecular weights of 14 and 6.5 kDa, respectively. c SDS-PAGE Coomassie gel of cross-linked JD1–82 and CTD170–232 reacted with either DMTMM only or DMTMM with ADH. This experiment was performed three independent times. d Contact map of ADH/DMTMM cross-links identified for JD1–82 (red), CTD170–232 (green), and full-length DnaJB8 (black). The axes are colored in red and green for JD and CTD, respectively. Cross-link pairs between JD–JD and CTD–CTD are shown in dashed boxes colored in red and green, respectively. e Histogram of the number of intra-domain cross-links that are consistent with cross-link chemistry geometry (“satisfied”) in the ensemble of 5000 models. One model satisfies 13 out of 14 possible cross-links identified in our experiments. Cross-links are mapped onto best matching CTD structural model (inset), shown in white cartoon representation. Sites of cross-link are shown as red or blue spheres, for D/E and K, respectively. Dashed yellow lines connect linked amino acid pairs. f Overlay of our DnaJB8 CTD model generated by ab initio ROSETTA (green) with the published DnaJB6bΔST CTD (salmon) (PDB ID: 6U3R). The CTD sequences of DNAJB8 and DNAJB6 are shown with each β-strand highlighted and conserved NG and DG turns in blue.
