Domain-specific spectroscopy of 5-hydroxytryptophan-containing variants ofEscherichia coli DnaJ

Tryptophan-containing variants of Escherichia coli DnaJ protein were constr ucted in order to examine the hypothetical domain structure by fluorescence quenching and denaturant-induced unfolding. Two residues in the J-domain a nd one in the Gly/Phe-rich region were targeted for replacement and the pro teins were expressed in a tryptophan auxotrophic strain in the presence of 5-hydroxytryptophan (5-HW). Fluorescence quenching with iodide of 5-HW in t he variant proteins suggests that the Gly/Phe-rich region is more accessibl e to solvent than the J-domain. This is consistent with the proposal that t he Gly/Phe-rich region is unstructured. Unfolding of the 5-HW-containing va riants was monitored by fluorescence, and the results showed that the unfol ding of the J-domain is cooperative and the unfolding of the Gly/Phe-rich r egion is not cooperative. (C) 2000 Elsevier Science B.V. All rights reserve d.