CHARACTERIZATION OF AMINO-ACID SIDE-CHAIN DYNAMICS IN A ZINC-FINGER PEPTIDE USING C-13 NMR-SPECTROSCOPY AND TIME-RESOLVED FLUORESCENCE SPECTROSCOPY

Proton-detected C-13 NMR spectroscopy has been used to measure spin-la ttice and spin-spin relaxation rate constants and the steady-state {H- 1}-C-13 nuclear Overhauser effect enhancements for the protonated arom atic carbon spins and for the methyl carbon spins in the zinc complex of a single zinc-finger peptide. Measurements were performed at 11.7 T for aromatic spins and at 7.0 and 11.7 T for methyl spins. Time-resol ved fluorescence spectroscopy has been used to measure the polarizatio n anisotropy decay for the single tyrosyl fluorophore in the peptide i n the presence and absence of zinc. The NMR relaxation and fluorescenc e depolarization data are analyzed using a model-free formalism in whi ch internal motions are characterized by order parameters and effectiv e correlation times. Order parameters for Tyr and His aromatic moietie s are similar to the order parameters for the corresponding backbone a lpha carbon spins reported previously (Palmer, A. G.; Rance, M.; Wrigh t, P. E. J. Am. Chem. Soc. 1991, 113, 4371-4380); thus, these aromatic side chains have little additional conformational freedom on picoseco nd to nanosecond times scales. In contrast, order parameters for Phe 1 0 indicate that the phenyl ring is more highly restricted than the bac kbone alpha carbon spin; thus, the order parameters reflect the packin g of the Phe 10 side chain in the hydrophobic core of the molecule. Ph enomenological spin-spin relaxation rate constants for the two His res idues provide evidence that a chemical-exchange process occurs in the zinc-binding site of the peptide. Order parameters for the symmetry ax es of the methyl groups are determined from the methyl C-H order param eters assuming tetrahedral geometry. In general, order parameters for the symmetry axes are smaller for residues with longer side chains; ho wever, the order parameters for Leu 16 have nearly maximal values and reflect the packing of Leu 16 in the hydrophobic core of the peptide. The order parameters for the symmetry axes of the geminal methyl group s of Val and Leu residues are compared to order parameters obtained pr eviously for the Val beta and Leu gamma methine carbon spins; the obse rved differences may reflect steric constraints on motions of the meth yl and methine groups. The symmetry axis order parameter for Ala 15 is larger than unity and indicates that relaxation is mediated by motion s with effective correlation times in excess of 100 ps. Analysis using specific models for internal motions of methyl groups suggests that t he motion of this methyl group cannot be described simply by restricte d rotational diffusion or three-site jump models. Order parameters mea sured by NMR and fluorescence spectroscopies for the tyrosyl ring in t he zinc complex of the peptide are in good agreement; consequently, or der parameters appear to be accurately determined by both experimental techniques. The overall rotational correlation time measured by fluor escence spectroscopy is 19% smaller than that measured by NMR spectros copy. Fluorescence depolarization is faster in the absence of zinc and probably is dominated by large amplitude local motions of the unfolde d peptide.