Domain-specific spectroscopy of 5-hydroxytryptophan-containing variants of Escherichia coli DnaJ.
Tryptophan-containing variants of Escherichia coli DnaJ protein were constructed in order to examine the hypothetical domain structure by fluorescence quenching and denaturant-induced unfolding. Two residues in the J-domain and one in the Gly/Phe-rich region were targeted for replacement and the proteins were expressed in a tryptophan auxotrophic strain in the presence of 5-hydroxytryptophan (5-HW). Fluorescence quenching with iodide of 5-HW in the variant proteins suggests that the Gly/Phe-rich region is more accessible to solvent than the J-domain. This is consistent with the proposal that the Gly/Phe-rich region is unstructured. Unfolding of the 5-HW-containing variants was monitored by fluorescence, and the results showed that the unfolding of the J-domain is cooperative and the unfolding of the Gly/Phe-rich region is not cooperative.
Greene
M K
MK
Department of Biochemistry (SL43), Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112-2699, USA.
Steede
N K
NK
Landry
S J
SJ
D006916:5-Hydroxytryptophan / Q000737:chemistry*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry*; D008958:Models, Molecular; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D006916:5-Hydroxytryptophan