Nuclease conA is a hybrid version of Staphylococcal nuclease that contains a six amino acid (β-turn substitute from concanavalin A. This hybrid protein has a much lower thermodynamic stability than does the wild-type protein. This enables the unfolding of the protein to be achieved easily by several types of perturbations. From temperature-, pressure-, and denaturant-induced unfolding studies, we have found the free energy change for unfolding, ΔG°un, to be approximately 1.4 kcal mo−1 at pH 7, 0.1 M NaCI, and 20 °C, as compared to a thermodynamic stability of approximately 5.5–6 kcal mo−1 for wild-type nuclease A. Due to its reduced thermodynamic stability, nuclease conA also shows evidence of unfolding at low-temperature (cold denaturation), with a temperature of maximum stability of 13–15 °C. The thermal unfolding of nuclease conA is shown to be two-state by simultaneous measurement of fluorescence and CD changes as a function of temperature, using a modified AVIV CD instrument. Increased hydrostatic pressure unfolds nuclease conA in what appears to be a two-state manner, with an apparent of ΔV°un approximately —100 ml mol−1. From studies of the pressure (p)-induced unfolding of this hybrid protein as a function of temperature (T), we can define the complete p-T free energy surface for the unfolding transition. In auxiliary studies, we have characterized the fluorescence intensity decay and anisotropy decay of the single tryptophan residue (Trp-140) of nuclease conA in the native state and in the unfolded state induced by temperature, pressure, and denaturant. For each type of perturbation, there is a red shift in fluorescence, a lowering of the mean fluorescence lifetime, and a lowering of the rotational correlation time of the tryptophan residue to a value of ~1 ns (compared to 10–15 ns for the native state). The thermodynamics of the unfolding of proteins has received renewed interest in recent years, owing to the availability of a rich variety of mutant proteins and to advances in our understanding of their structural features. Among the questions being asked are, What are the relative energetic contributions of the hydrophobic effect and other interaction forces?
Modification of the Single Tryptophan Residue of Staphylococcal Nuclease by a New Mild Oxidizing Agent