Perchlorate-induced conformational transition of Staphylococcal nuclease: evidence for an equilibrium unfolding intermediate.
The sodium perchlorate-induced conformational transition of Staphylococcal nuclease has been monitored by both circular dichroism (CD) and fluorescence spectroscopy. The perchlorate-induced transition is cooperative as observed by both spectroscopic signals. However, the protein loses only about one-third of its native far-UV CD signal at high perchlorate concentrations, indicating that a significant amount of secondary structure remains in the post-transition state. The remaining CD signal can be further diminished in a cooperative manner by the addition of the strong denaturant, urea. Near-UV CD spectra clearly show that the protein loses its tertiary structure in the perchlorate-induced denatured state. The perchlorate-induced transition curves were fit to the standard two-state model and the standard free energy change and m value of the transition are 2.3kcal/mol and 1.8kcal/(molM), respectively. By comparison, the urea-induced unfolding of Staphylococcal nuclease (in the absence of perchlorate) yields an unfolding free energy change, DeltaG(0,un), of 5.6kcal/mol and an m value of 2.3kcal/(molM). Thus, the thermodynamic state obtained in the post-transition region of perchlorate-induced conformation transition has a significantly lower free energy change, a high content of secondary structure, and diminished tertiary structure. These results suggest that the perchlorate-induced denatured state is a partially folded equilibrium state. Whether this intermediate is relevant to the folding/unfolding path under standard conditions is unknown at this time.
Maity
Haripada
H
Department of Chemistry and Biochemistry, University of Mississippi, University, MS 38677, USA.
Eftink
Maurice R
MR
D002942:Circular Dichroism; D007700:Kinetics; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000378:metabolism; D010472:Perchlorates / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D017670:Sodium Compounds / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013210:Staphylococcus / Q000201:enzymology*; D014508:Urea / Q000378:metabolism
D010472:Perchlorates; D017670:Sodium Compounds; D014508:Urea; C031068:sodium perchlorate; D008836:Micrococcal Nuclease