INTRAMOLECULAR MOTIONS OF A ZINC FINGER DNA-BINDING DOMAIN FROM XFIN CHARACTERIZED BY PROTON-DETECTED NATURAL ABUNDANCE C-12 HETERONUCLEAR NMR-SPECTROSCOPY

The zinc finger DNA-binding domain Xfin-31 is a 25-residue peptide that binds a single zinc atom and forms a compact globular structure in solution. To characterize the intramolecular dynamics of Xfin-31, the C-13 spin-lattice and spin-spin relaxation rate constants and the {H-1}-C-13 nuclear Overhauser effect (NOE) enhancements have been measured for the backbone and side chain methine carbons by two-dimensional proton-detected heteronuclear NMR spectroscopy at a C-13 Larmor frequency of 125 MHz and natural C-13 abundance. The relaxation rate constants and the NOE enhancements have been analyzed by using a model-free formalism that depends on the overall rotational correlation time of the molecule, tau-m, the order parameter S, and effective internal correlation time, tau-e, for each methine carbon. The optimized global value of tau-m is 1.88 +/- 0.02 ns. The backbone C-alpha carbons are grouped into four categories based on the values of the order parameters: the N-terminal residue Tyr1 with S2 = 0.73 +/- 0.04; the C-terminal residues Lys24 and Asn25 with S2 < 0.5; residues Phe10-Lysl3 with an average S2 = 0.77 +/- 0.03; and the remainder of the backbone carbon nuclei with an average S2 = 0.89 +/- 0.05. For the side chain C-beta of Val11 and Val22 and the C-gamma of Leu5, the values of S2 are 0.62 +/- 0.03, 0.66 +/- 0.04, and 0.47 +/- 0.03, respectively. Estimates of tau-e could be obtained for 13 of the backbone and 3 of the side chain methine carbons. Excluding the terminal residues, the average value of tau-e for the backbone carbon nuclei was 34 +/- 16 ps. With the exception of the terminal residues, the motions of the backbone carbon nuclei of Xfin-31 are highly restricted. Residues 10-13, which form a turn between the beta-sheet and the helix present in the three-dimensional structure of Xfin-31, have a slightly higher mobility than the rest of the interior backbone, and the two residues at the C-terminus have considerable conformational flexibility. The side chains of the hydrophobic Val and Leu residues are more mobile than the backbone but are still significantly restricted, which indicates that Xfin-31 is compact despite its small size. Systematically large spin-spin relaxation rates for residues in the zinc binding site imply that conformational exchange may occur in this region on a time scale longer than the overall rotational correlation time.