Effects of guanidine hydrochloride on the conformation and enzyme activity of streptomycin adenylyltransferase monitored by circular dichroism and fluorescence spectroscopy.
Equilibrium denaturation of streptomycin adenylyltransferase (SMATase) has been studied by CD spectroscopy, fluorescence emission spectroscopy, and binding of the hydrophobic dye 1-anilino-8-naphthalene sulfonic acid (ANS). Far-UV CD spectra show retention of 90% native-like secondary structure at 0.5 M guanidine hydrochloride (GdnHCl). The mean residue ellipticities at 222 nm and enzyme activity plotted against GdnHCl concentration showed loss of about 50 and 75% of secondary structure and 35 and 60% of activity at 0.75 and 1.5 M GdnHCl, respectively. At 6 M GdnHCl, there was loss of secondary structure and activity leading to the formation of GdnHCl-induced unfolded state as evidenced by CD and fluorescence spectroscopy as well as by measuring enzymatic activity. The denaturant-mediated decrease in fluorescence intensity and 5 nm red shift of lambda(max) point to gradual unfolding of SMATase when GdnHCl is added up from 0.5 M to a maximum of 6 M. Decreasing of ANS binding and red shift (approximately 5 nm) were observed in this state compared to the native folded state, indicating the partial destruction of surface hydrophobic patches of the protein molecule on denaturation. Disruption of disulfide bonds in the protein resulted in sharp decrease in surface hydrophobicity of the protein, indicating that the surface hydrophobic patches are held by disulfide bonds even in the GdnHCl denatured state. Acrylamide and potassium iodide quenching of the intrinsic tryptophan fluorescence of SMATase showed that the native protein is in folded conformation with majority of the tryptophan residues exposed to the solvent, and about 20% of them are in negatively charged environment.
Jana
Snehasis
S
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology-Delhi, Hauz Khas, New Delhi 110016, India.
Chaudhuri
Tapan Kumar
TK
Deb
J K
JK
D020106:Acrylamide / Q000494:pharmacology; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D002942:Circular Dichroism; D019791:Guanidine / Q000494:pharmacology*; D009169:Mycobacterium tuberculosis / Q000201:enzymology; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism*; D011193:Potassium Iodide / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D011193:Potassium Iodide; D020106:Acrylamide; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D009713:Nucleotidyltransferases; C022678:streptomycin 3''-adenylyltransferase; D019791:Guanidine