The denatured "state" of a protein is a distribution of many different molecular conformations, the averages of which are measured by experiments. The properties of this ensemble depend sensitively on the solution conditions. There is now considerable evidence that even in strong denaturants such as 6M GuHC1 and 9M urea, some structure may remain in protein chains. Under milder or physiological conditions, the denatured states of most proteins appear to be highly compact with extensive secondary structure. Both theoretical and experimental studies suggest that hydrophobic interactions, chain conformational entropies, and electrostatic forces are dominant in determining this structure. The denaturation reaction of many proteins in GuHC1 or urea can be most simply modelled as a two-state transition between the native structure and a relatively compact denatured state, which then undergoes a gradual increase in radius on further addition of denaturant. However, when a protein acquires a large net charge in acids or bases, it can have two stable denatured populations, one compact and the other more highly unfolded. The prediction and elucidation of the structural details of the non-native states of proteins may ultimately prove to be as difficult as predicting the native structures, particularly for D0, the denatured state under physiological conditions. Just as with the native state, the structure of this biologically important denatured state appears to depend on the amino acid sequence. The development of synthetic, peptide and protein fragment models of the denatured state and the recent progress in NMR spectroscopy provide bases for optimism that new insights will be gained into this poorly understood realm of protein biochemistry.

Experimental methods have demonstrated that when a protein unfolds, not all of its structure is lost. Here we report measurement of residual dipolar couplings in denatured forms of the small protein staphylococcal nuclease oriented in strained polyacrylamide gels. A highly significant correlation among the dipolar couplings for individual residues suggests that a native-like spatial positioning and orientation of chain segments (topology) persists to concentrations of at least 8 molar urea. These data demonstrate that long-range ordering can occur well before a folding protein attains a compact conformation, a conclusion not anticipated by any of the standard models of protein folding.

To quantitate the contributions of the large hydrophobic residues in staphylococcal nuclease to the stability of its native state, single alanine and glycine substitutions were constructed by site-directed mutagenesis for each of the 11 leucine, 9 valine, 7 tyrosine, 5 isoleucine, 4 methionine, and 3 phenylalanine residues. In addition, each isoleucine was also mutated to valine. The resulting collection of 83 mutant nucleases was submitted to guanidine hydrochloride denaturation using intrinsic tryptophan fluorescence to monitor the equilibrium constant between the native and denatured states. From analysis of these data, each mutant protein's stability to reversible denaturation (delta GH2O) and sensitivity to guanidine hydrochloride (mGuHCl or d(delta G)/d[GuHCl]) were obtained. Four unexpected trends were observed. (1) A striking bipartite distribution was found for sites of mutations that altered mGuHCl: mutations that increased this parameter only involved residues that contribute side chains to the major hydrophobic core centered around a five-strand beta-barrel, whereas mutations that caused mGuHCl to decrease clustered around a second, smaller and less well-defined hydrophobic core. (2) The average stability loss for mutants in each of the six residue classes was 2-3 times greater than that estimated on the basis of the free energy of transfer of the hydrophobic side chain from water to n-octanol. (3) The magnitude of the stability loss on substituting Ala or Gly for a particular type of amino acid varied extensively among the different sites of its occurrence in nuclease, indicating that the environment surrounding a specific residue determines how large a stability contribution its side chain will make.(ABSTRACT TRUNCATED AT 250 WORDS)

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Eleven mutant forms of staphylococcal nuclease with one or more defined amino acid substitutions have been analyzed by solvent denaturation by using intrinsic fluorescence to follow the denaturation reaction. On the basis of patterns observed in the value of m--the rate of change of log Kapp (the apparent equilibrium constant between the native and denatured states) with denaturant concentration--these proteins can be grouped into two classes. For class I mutants, the value of m with guanidine hydrochloride is less than the wild-type value and is either constant or increases slightly with increasing denaturant; the value of m with urea is also less than wild type but shows a marked increase with increasing denaturant concentration, often approaching but never exceeding the wild-type value. For class II mutants, m is constant and is greater than wild type in both denaturants, with the increase being consistently larger in guanidine hydrochloride than in urea. When double or triple mutants are constructed from members of the same mutant class, the change in m is usually the sum of the changes produced by each mutation in isolation. One plausible explanation for these altered patterns of denaturation is that chain-chain or chain-solvent interactions in the denatured state have been modified--interactions which appear to involve hydrophobic groups.

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Experimental studies of the physical interactions that stabilize protein structure are complicated by the fact that proteins do not unfold to a simple reference state. When their folded structure breaks down, protein chains do not become random coils. Instead, they enter a poorly understood ensemble of partially folded states known collectively as the denatured state. Although it has long been held that agents that promote protein unfolding act specifically on the denatured state, the idea that mutations can exert their destabilizing (or in some cases, stabilizing) effects directly on this state is not widely accepted. A large body of thermodynamic data on mutant proteins plus a limited amount of structural information describing mutational effects on denatured states indicate that 1) the denatured state plays a central role in all aspects of protein stability, including mutant effects, and 2) a quantitative understanding of how amino acid sequence encodes protein structure will probably depend on a more complete picture of this complex, difficult-to-study state.

delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni promotes the highly efficient isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by means of a direct and stereospecific transfer of the 4 beta-proton to the 6 beta-position, via an enolic intermediate. An acidic residue responsible for the protonation of the 3-carbonyl function of the steroid and a basic group concerned with the proton transfer have been implicated in the catalytic mechanism. Recent NMR studies with a nitroxide spin-labeled substrate analogue have allowed positioning of the steroid into the 2.5-A X-ray crystal structure of the enzyme [Kuliopulos, A., Westbrook, E.M., Talalay, P., & Mildvan, A.S. (1987) Biochemistry 26, 3927-3937], thereby corroborating the approximate location of the steroid binding site deduced from a difference Fourier X-ray diffraction map of the 4-(acetoxymercuri)estradiol-isomerase complex [Westbrook, E.M., Piro, O.E., & Sigler, P.B. (1984) J. Biol. Chem. 259, 9096-9103]. The steroid lies in a hydrophobic cavity near Asp-38, Tyr-14, and Tyr-55. In order to assess the role of these amino acid residues in catalysis, the gene for isomerase was cloned, sequenced, and overexpressed in Escherichia coli [Kuliopulos, A., Shortle, D., & Talalay, P. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8893-8897], and the following mutants were prepared: Asp-38 to asparagine (D38N) and Tyr-14 and Tyr-55 to phenylalanine (Y14F and Y55F, respectively). The kcat value of the D38N mutant enzyme is 10(5.6)-fold lower than that of the wild-type enzyme, suggesting that Asp-38 functions as the base which abstracts the 4 beta-proton of the steroid in the rate-limiting step. Threefold lower Km values in all mutants indicate tighter binding of the substrate to the more hydrophobic sites. In comparison with the wild-type enzyme, the Y55F mutant shows only a 4-fold decrease in kcat while the Y14F mutant shows a 10(4.7)-fold decrease in kcat, suggesting that Tyr-14 is the general acid. The red shift of the ultraviolet absorption maximum of the competitive inhibitor 19-nortestosterone from 248 to 258-260 nm, which occurs upon binding to the wild-type enzyme [Wang, S.F., Kawahara, F.S., & Talalay, P. (1963) J. Biol. Chem. 238, 576-585], is mimicked in strong acid. This spectral shift was also observed with the D38N and Y55F mutants, but not on binding of the steroid to the Y14F mutant.(ABSTRACT TRUNCATED AT 400 WORDS)