Denatured state structure

Wrabl, J. and D. Shortle. 1999. A model of the changes in denatured state structure underlying m value effects in staphylococcal nuclease. Nature Structural Biology. 6, 876. 

Bond C ], K-B Wong, ] Clarke, A R Ferscht and V Daggett 1997. Characterisation of Residual Structure in the Tliermally Denatured State of Barnase by Simulation and Experiment Description of the Folding Pathway. Proceedings of the National Academy of Sciences USA 94 13409-13413. 

Shortle has focused on the unfolded state for more than a decade, leading up to his recent demonstration using residual dipolar couplings that staphylococcal nuclease retains global structure in 8 M urea. His chapter on The Expanded Denatured State sets the stage. Dunker etal. then explore the complementary world of disordered regions within... 

Toward a Taxonomy of the Denatured State Small Angle Scattering Studies of Unfolded Proteins by Millett et al. assesses denatured states induced by heat, cold, and solvent for evidence of residual structure, while Insights into the Structure and Dynamics of Unfolded Proteins from NMR by Dyson and Wright describes their extensive investigations of residual structure in the unfolded state. 

Because the denatured state has long been thought to be essentially random and because of the inherent difficulties in defining and interpreting structural data averaged in very complex ways, protein chemists have been slow to take up the structural characterization of denatured proteins. Yet a more complete description of the relatively small amounts of persistent structure they display is not simply of academic interest. There are compelling reasons to pursue structural studies of proteins that are not folded. 

First, proteins refold from the denatured state, not from the hypothetical random coil state. It is the starting point of all refolding reactions, whether in a cell or in a test tube. To understand any chemical reaction, structural features of the reactant and the product must be compared to quantify the changes that occur, for these changes define the reaction. 

In this review, NMR analysis of the denatured state of staphylococcal nuclease is briefly reviewed in nontechnical language. Most of the work has come from the author s laboratory over the past eight years. The initial experiments, which only measure local structural parameters, reported small amounts of persisting helical structure, two turns, and indirect evidence for perhaps a three-strand beta meander. When applied to the denatured state in 6 M urea, the same experiments indicated that most of these features are lost. 

However, recent application of two types of NMR methodologies that provide long-range structural information have painted a very different picture. From these experiments, the denatured state in 0 M urea and 8 M urea appear to be highly similar, both retaining the same overall spatial positioning and orientation of the chain seen in the folded conformation. 

Fig. 1. Schematic diagram of nuclease A131A in the folded conformation. The alpha helices and beta strands are labeled. NMR analysis suggests the two turns and one helix in black are modestly populated in the denatured state, whereas the shaded helix is slightly populated. Strands / l-/ 2-/ 3 form an extended structure about which littie is known. Reproduced from Barron, L. D., Hecht, L., Blanch, E. W., and Bell, A. F. (2000). Prog. Biophys. Mol Chem. 73, 1-49. 2000, with permission from Elsevier Science.

The most important feature of the information in dipolar couplings is that it is independent of distance. The data can be envisioned as reflecting the relative orientation of pairs of bond vectors, with the intervening distance having no effect (Meiler et al., 2000). Thus dipolar couplings can potentially provide a method for characterizing the structure of denatured proteins, provided that the denatured state ensemble of conformations retains several levels of nonrandomness. 

The changes in structure of denatured nuclease as a function of urea concentration (Fig. 3) suggest that, as hydrophobic interactions are weakened and the backbone becomes more highly solvated, the chain expands gradually. The data presented by Millet et al. in this volume suggest that this expansion does not continue asymptotically as predicted by simple polymer physical chemistry. This is the behavior expected for a polypeptide chain trapped in a small region of conformation space. Most, perhaps all, of the conformations accessible in the expanded denatured state may have a native-like topology. 

Fig. 7. Simple schematic diagram representing three conformational states of proteins The expanded denatured state whose structure is determined by steric interactions the compact denatured state, with structure determined by hydrophobic buril and the native state, with structure determined by dispersion forces, hydrogen bonds, and electrostatics.

IV. Reconciling the Random Coil with a Structured Denatured State. 257... 

The cold-denatured state clearly has residual structure in that it is collapsed and exhibits a fairly globular Kratky scattering profile. Truncated, equilibrium unfolded states appear similarly compact (and have been shown by other spectroscopic means to have structure). But what of the highly expanded, urea, GuHCl, or thermally denatured states ... 

In contrast to the reconciliation of short-range structure with net random coil-like behavior, the presence of long-range order in even the most highly denatured states is perhaps more problematic (Shortle and... 

Results from thermal denaturation and heat capacity studies have shown that the proteins are not necessarily completely unfolded in this process. The volume observations also suggest that the denatured state is not one in which all hydrophobic groups are exposed to water. But the results can also be understood from the effect of close polar and electrostatic groups interacting with the water structure surrounding the hydrophobic groups. The volume change is heavily... 

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