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Published August 28, 2001 | public
Journal Article

Polar residues in the protein core of Escherichia coli thioredoxin are important for fold specificity


Most globular proteins contain a core of hydrophobic residues that are inaccessible to solvent in the folded state. In general, polar residues in the core are thermodynamically unfavorable except when they are able to form intramolecular hydrogen bonds. Compared to hydrophobic interactions, polar interactions are more directional in character and may aid in fold specificity. In a survey of 263 globular protein structures, we found a strong positive correlation between the number of polar residues at core positions and protein size. To probe the importance of buried polar residues, we experimentally tested the effects of hydrophobic mutations at the five polar core residues in Escherichia coli thioredoxin. Proteins with single hydrophobic mutations (D26I, C32A, C35A, T66L, and T77V) all have cooperative unfolding transitions like the wild type (wt), as determined by chemical denaturation. Relative to wt, D26I is more stable while the other point mutants are less stable. The combined 5-fold mutant protein (IAALV) is less stable than wt and has an unfolding transition that is substantially less cooperative than that of wt. NMR spectra as well as amide deuterium exchange indicate that IAALV is likely sampling a number of low-energy structures in the folded state, suggesting that polar residues in the core are important for specifying a well-folded native structure.

Additional Information

© 2001 American Chemical Society. Received March 1, 2001; Revised Manuscript Received June 5, 2001. Publication Date (Web): August 1, 2001. This research was supported by the Howard Hughes Medical Institute and the Ralph M. Parsons Foundation (S.L.M.), the Helen G. and Arthur McCallum Foundation, the Evelyn Sharp Graduate Fellowship, and Grant GM07616 from the National Institutes of Health (D.N.B.). We thank Scott Ross for guidance and assistance with NMR studies as well as useful discussions, and Arthur Street for compiling the data set analyzed in Figure 1.

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