Perchlorate-induced conformational transition of Staphylococcal nuclease: evidence for an equilibrium unfolding intermediate

• Published in: Archives of biochemistry and biophysics 2004-11, Vol.431 (1), p.119-123
• Main Authors: Maity, Haripada, Eftink, Maurice R.
• Format: Article
• Language: English
• Subjects: Chaotropic salts; Circular Dichroism; Hoffmeister series; Kinetics; Micrococcal Nuclease - chemistry; Micrococcal Nuclease - metabolism; Molten globule; Perchlorate-induced denatured state; Perchlorates - metabolism; Protein Conformation; Protein Denaturation; Protein folding; Sodium Compounds - metabolism; Spectrometry, Fluorescence; Staphylococcal nuclease; Staphylococcus - enzymology; Urea - metabolism
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2004.07.023

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 2.3 kcal/mol and 1.8 kcal/(mol M), respectively. By comparison, the urea-induced unfolding of Staphylococcal nuclease (in the absence of perchlorate) yields an unfolding free energy change, Δ G 0,un, of 5.6 kcal/mol and an m value of 2.3 kcal/(mol M). 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.