Association of fluorescent probes 1-anilinonaphthalene-8-sulfonate and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid with T7 RNA polymerase.
T7 RNA polymerase is an enzyme that carries out transcription using DNA as the template and ribonucleotides as the substrates. Here we report the association of the polymerase with 1-anilinonaphthalene-8-sulfonate (ANS) and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS), which are two fluorescent hydrophobic probes that are frequently used to study structural perturbations in proteins and intermediate states of proteins during folding and unfolding. Our results from the fluorescence titration data show that these two molecules bind to the enzyme with dissociation constants on the micromolar order. The results from the tryptic digestion of the enzyme in the absence and presence of the probes show that they inhibit the rate of tryptic digestion. Circular dichroism spectroscopic studies of the protein in the near UV region indicate that both probes induce tertiary structural changes in the polymerase. There is also a probe (ANS or bis-ANS) induced inhibition of the enzymatic activity. All these results are attributed to association of the probes with the enzyme, leading to an alteration in the conformation of T7 RNA polymerase. This limits the use of these extrinisic probes to the study of the folding properties of the enzyme.
Ghosh
Utpal
U
Biophysics Division, Saha Institute of Nuclear Physics, 37 Belgachia Road, Kolkata 700 037, India.
Das
Mili
M
Dasgupta
Dipak
D
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D009282:Naphthalenesulfonates / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014764:Viral Proteins
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D014764:Viral Proteins; C027132:1-anilino-8-naphthalenesulfonate; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases