Conformational changes at the active site of bovine pancreatic RNase A at low concentrations of guanidine hydrochloride probed by pyridoxal 5'-phosphate.
The alpha-amino group of Lys-1 and the epsilon-amino groups of Lys-41 and Lys-7 were labeled with pyridoxal 5'-phosphate (PLP) separately. The effects of GdnHCl on the activities and the fluorescence properties of the derivatives were compared. Both the fluorescence intensity and anisotropy of the probe introduced at the active site Lys-41 decreased obviously during denaturation by low concentrations of GdnHCl indicating conformational change of the active site is a parallel event to the inactivation of the enzyme. Time-correlated fluorescence lifetime measurements revealed the existence of two conformational states of PLP-modified RNase A and a shift of the conformation in favor of the slow-decay component with increasing GdnHCl concentration. The decrease in activity of RNase A at low concentrations of GdnHCl is therefore due to partial unfolding of the molecule, particularly at the active site. The experimental results of this work support the suggestion that the conformation at the active site is more easily perturbed and hence more flexible than the molecule as a whole.
Xiao
G S
GS
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing, China.
Zhou
J M
JM
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005454:Fluorescence Polarization; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D007700:Kinetics; D015335:Molecular Probes / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014443:Tyrosine / Q000378:metabolism
D006146:Guanidines; D015335:Molecular Probes; D014443:Tyrosine; D011732:Pyridoxal Phosphate; D012259:Ribonuclease, Pancreatic; D019791:Guanidine