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Solution structure of a protein denatured state and folding intermediate

Abstract

The most controversial area in protein folding concerns its earliest stages. Questions such as whether there are genuine folding intermediates, and whether the events at the earliest stages are just rearrangements of the denatured state1 or progress from populated transition states2, remain unresolved. The problem is that there is a lack of experimental high-resolution structural information about early folding intermediates and denatured states under conditions that favour folding because competent states spontaneously fold rapidly. Here we have solved directly the solution structure of a true denatured state by nuclear magnetic resonance under conditions that would normally favour folding, and directly studied its equilibrium and kinetic behaviour. We engineered a mutant of Drosophila melanogaster Engrailed homeodomain that folds and unfolds reversibly just by changing ionic strength. At high ionic strength, the mutant L16A is an ultra-fast folding native protein, just like the wild-type protein; however, at physiological ionic strength it is denatured. The denatured state is a well-ordered folding intermediate, poised to fold by docking helices and breaking some non-native interactions. It unfolds relatively progressively with increasingly denaturing conditions, and so superficially resembles a denatured state with properties that vary with conditions. Such ill-defined unfolding is a common feature of early folding intermediate states and accounts for why there are so many controversies about intermediates versus compact denatured states in protein folding.

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Figure 1: Structure of En-HD L16A at low ionic strength and summary of sequential NOEs and secondary chemical shift values (Protein Data Bank code 1ZTR).
Figure 2: Backbone dynamics of wild-type and En-HD L16A protein.
Figure 3: Effects of salt on the properties of wild-type En-HD and L16A, and the difficulties of distinguishing between cooperative and non-cooperative transitions.
Figure 4: Relaxation kinetics of wild-type En-HD and the L16A mutant at various concentrations of NaCl as a function of temperature at 50 mM NaAc, pH 5.7.

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Acknowledgements

T.L.R. was supported by a Trinity College Internal Graduate Studentship, and J.S.M. by a Gates Cambridge Scholarship. We would like to thank T. J. Rutherford and L. Kowalik for help with NMR, and J. M. Perez Canadillas for help with protein expression.Author Contributions T.L.R. was responsible for the major experimental work and planning of this study, with further contributions from J.S.M., U.M. and S.M.V.F. A.R.F. wrote the paper with T.L.R. and performed data analysis.

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Supplementary information

Supplementary Figure S1

HN-N RDCs for L16A at 298 K (calculated as D – J coupling). A: 9% acrylamide. B: 12.5% Pf1 phage at pH 7.0. (JPG 21 kb)

Supplementary Notes

This file contains Supplementary Methods and Supplementary Tables S1 and S2. (DOC 45 kb)

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Religa, T., Markson, J., Mayor, U. et al. Solution structure of a protein denatured state and folding intermediate. Nature 437, 1053–1056 (2005). https://doi.org/10.1038/nature04054

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