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Water and urea interactions with the native and unfolded forms of a ß-barrel protein

¹ Department of Biophysical Chemistry, Lund University, SE-22100 Lund, Sweden
² Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, Minnesota 55905, USA

Reprint requests to: Bertil Halle, Department of Biophysical Chemistry, Lund University, Box 124, SE-22100 Lund, Sweden; e-mail: bertil.halle{at}bpc.lu.se; fax: 46-46-222-4543.

A fundamental understanding of protein stability and the mechanism of denaturant action must ultimately rest on detailed knowledge about the structure, solvation, and energetics of the denatured state. Here, we use ¹⁷O and ²H magnetic relaxation dispersion (MRD) to study urea-induced denaturation of intestinal fatty acid-binding protein (I-FABP). MRD is among the few methods that can provide molecular-level information about protein solvation in native as well as denatured states, and it is used here to simultaneously monitor the interactions of urea and water with the unfolding protein. Whereas CD shows an apparently two-state transition, MRD reveals a more complex process involving at least two intermediates. At least one water molecule binds persistently (with residence time >10 nsec) to the protein even in 7.5 M urea, where the large internal binding cavity is disrupted and CD indicates a fully denatured protein. This may be the water molecule buried near the small hydrophobic folding core at the D–E turn in the native protein. The MRD data also provide insights about transient (residence time <1 nsec) interactions of urea and water with the native and denatured protein. In the denatured state, both water and urea rotation is much more retarded than for a fully solvated polypeptide. The MRD results support a picture of the denatured state where solvent penetrates relatively compact clusters of polypeptide segments.

Keywords: Protein denaturation; fatty acid-binding protein; urea; solvent exchange; magnetic relaxation dispersion

Abbreviations: CD, circular dichroism • I-FABP, intestinal fatty acid-binding protein • MRD, magnetic relaxation dispersion • NOE, nuclear Overhauser effect

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