Effects of Mg2+ and denaturants on the unfolding pattern of DNA-T--a replication protein of E. coli.
Escherichia coli-DNA-T protein is a key component of a multiprotein complex called the primosome which is involved in the initiation of DNA replication. The thermal and urea induced unfolding transition of this protein in the presence and absence of Mg2+ was studied using circular dichroism (CD) and fluorescence spectroscopy as probes. Quenching of the intrinsic fluorescence of DNA-T was observed in the thermal unfolding while formation of a hyperfluorescent form of the protein was found in the urea induced unfolding process. The CD studies showed a monophasic transition curve for thermal unfolding in the presence and absence of Mg2+. Biphasic curves indicative of the formation of intermediates was observed in the urea induced unfolding. The results suggest that the pathways of unfolding of thermal- and urea-induced transitions are different. MgCl2, which affects the conformation of the protein and stabilises the secondary structure, also affects the unfolding pattern.
Antony
T
T
Centre for Biochemical Technology, Delhi, India.
Kumar
S
S
Chauhan
M
M
Atreyi
M
M
Khatri
G S
GS
D001426:Bacterial Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D002942:Circular Dichroism; D004261:DNA Replication; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D005453:Fluorescence; D008274:Magnesium / Q000737:chemistry* / Q000494:pharmacology; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D013696:Temperature; D014508:Urea / Q000737:chemistry
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C104198:DnaT protein, E coli; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D014508:Urea; D008274:Magnesium