Staphylococcal nuclease refolding

Panick, G., Malessa, R., Winter, R., Rapp, G., Frye, K.J., Royer, C.A. Structural characterization of the pressure-denaturated state and unfolding/refolding kinetics of staphylococcal nuclease by synchrotron small-angle X-ray scattering and Fourier-transform infrared spectroscopy, J. Mol. Biol. 275 (1998) 389-402. 

S. Patel, P. Sista, P. V. Balaji, and Y. U. Sasidhar,/. Mol. Graph. Model., 25, 103 (2006). -Hairpins with Native-Like and Non-Native Hydrogen Bonding Patterns Could Form During the Refolding of Staphylococcal Nuclease. 

Despite the scarcity of the data, for several of these proteins, common features appear in the unfolding-folding process, particularly the reversibility of the process. The existence of intermediates was deduced for only few of them. The ability of a polypeptide chain to refold after the removal of a short peptide was studied extensively only for staphylococcal nuclease. 

The regaining of activity by a protein without disulfide bridges can be a rather rapid process. For staphylococcal nuclease, the overall rate of refolding is rather rapid. The halftime for folding is 0.1-0.2 sec. Cytochrome c... 

Staphylococcal nuclease undergoes a reversible transition between pH 3 and 4. Kinetics of refolding of the acidified enzyme was studied by a stopped-flow method by measuring the increase of fluorescence of Trp 140 (Schechter et ai, 1970 Epstein et ai, 1971a). Biphasic kinetics were observed as shown in Fig. 7.5. Two first-order processes correctly describe the kinetics with rate constants of 12.4 and 1.9 sec , respectively for fast and slow processes. The two processes are not so broadly separated in the time scale as is the... 

Fig. 7.5. Biphasic kinetics for refolding of staphylococcal nuclease denatured at low pH, observed by variation of fluorescence of Trp 140. Initial pH values ( ) 3.27 (O) 3.45 (A) 3.65 (A) 3.80. Final pH was 6.7 in all experiments (from Epstein et aL, 1971a),...

Fig. 7.6. Variations with temperature of the two rate constants observed for refolding of staphylococcal nuclease. Activation entropy for the slower process was 33 eu, and for the faster one 58 eu (from Epstein et aU 1971a).

Since antibody probes are extremely sensitive to variations, even subtle ones, in protein conformation, they are potent but rather delicate tools for the study of protein folding or unfolding. For the first time, Anfinsen s group developed a quantitative immunochemical approach to study the refolding of staphylococcal nuclease (Sachs et aL, 1972a,b,c, 1974 Eastlake et ai, 1974 Furie et aL, 1974, 1975). From that, different methods using specific antibodies for detection and characterization of intermediates in protein... 

An immunochemical approach was used to study conformational equilibria of isolated polypeptide fragments as well as entire staphylococcal nuclease (Sachs et al, 1974 Furie et al, 1975). An antibody population directed against the unfolded form of fragments (99-126)r was isolated to study the equilibrium between unfolded nuclease and the spontaneously refolded form. Figure 9.8 is a schematic representation of the presumed two-state equilibrium between native and unfolded forms. It may be described by the following expression ... 

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