This chapter describes the studies of staphylococcal nuclease that provide compelling evidence that (1) the denatured state of this protein under many conditions is not a random coil-it has residual structure; (2) this residual structure can be dramatically altered by single mutations; and (3) yet for most mutations, the free energy difference between N and D is changed by only a few kilocalories. As measured by a “free energy distance,” the D state is not far away from the N state. These observations suggest that the distribution of D microstates for wild-type nuclease is very broad and that mutations act by shifting the distribution along a number of different structural axes, presumably in directions that are, on average, unfavorable for refolding. The m-value effects are the most obvious manifestation of these shifts in microstate populations: changes in stability, enthalpy, entropy, and heat capacity must also accompany these changes in the D state.
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