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Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106-4935, USA
(RECEIVED May 14, 2004; FINAL REVISION August 10, 2004; ACCEPTED August 10, 2004)
The classical Zn finger contains a phenylalanine at the crux of its three architectural elements: a 
-hairpin, an 
-helix, and a Zn2+-binding site. Surprisingly, phenylalanine is not required for high-affinity Zn2+ binding, but instead contributes to the specification of a precise DNA-binding surface. Substitution of phenylalanine by leucine leads to a floppy but native-like structure whose Zn affinity is maintained by marked entropy–enthalpy compensation (
H –8.3 kcal/mol and –T S 7.7 kcal/mol). Phenylalanine and leucine differ in shape, size, and aromaticity. To distinguish which features correlate with dynamic stability, we have investigated a nonstandard finger containing cyclohexanylalanine at this site. The structure of the nonstandard finger is similar to that of the native domain. The cyclohexanyl ring assumes a chair conformation, and conformational fluctuations characteristic of the leucine variant are damped. Although the nonstandard finger exhibits a lower affinity for Zn2+ than does the native domain ( G –1.2 kcal/mol), leucine-associated perturbations in enthalpy and entropy are almost completely attenuated ( H –0.7 kcal/mol and –T S –0.5 kcal/mol). Strikingly, global changes in entropy (as inferred from calorimetry) are in each case opposite in sign from changes in configurational entropy (as inferred from NMR). This seeming paradox suggests that enthalpy–entropy compensation is dominated by solvent reorganization rather than nominal molecular properties. Together, these results demonstrate that dynamic and thermodynamic perturbations correlate with formation or repair of a solvated packing defect rather than type of physical interaction (aromatic or aliphatic) within the core.
Keywords: NMR spectroscopy; isothermal titration calorimetry; protein design; nonstandard mutagenesis; structural water
Abbreviations: CD, circular dichroism • Cyc, cyclohexanylalanine • DG, distance geometry • EEC, entropy–enthalpy compensation • ITC, isothermal titration calorimetry • MD, molecular dynamics • NMR, nuclear magnetic resonance • NOE, nuclear Overhauser enhancement • NOESY, NOE spectroscopy • RMD, restrained molecular dynamics • RMS; root-mean-square • RMSD, RMS difference. Natural amino acids are designated by standard single- and three-letter codes • 2D, two-dimensional • 3D, three-dimensional
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04866404.
Reprint requests to: Michael A. Weiss, Department of Biochemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4935, USA; e-mail: michael.weiss{at}case.edu; fax: (216) 368-3419.
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