Characterization of amino acid side chain dynamics in a zinc-finger peptide using carbon-13 NMR spectroscopy and time-resolved fluorescence spectroscopy

Palmer, A.G.I.i; Hochstrasser, R.A.; Millar, D.P.; Rance, M.; Wright, P.E.

Journal of the American Chemical Society 115(14): 6333-6345

1993


ISSN/ISBN: 0002-7863
DOI: 10.1021/ja00067a057
Accession: 008295638

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Abstract
Proton-detected 13C NMR spectroscopy has been used to measure spin-lattice and spin-spin relaxation rate constants and the steady-state (-1H)-13C nuclear Overhauser effect enhancements for the protonated aromatic 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-resolved fluorescence spectroscopy has been used to measure the polarization anisotropy decay for the single tyrosyl fluorophore in the peptide in the presence and absence of zinc. The NMR relaxation and fluorescence depolarization data are analyzed using a model-free formalism in which internal motions are characterized by order parameters and effective correlation times. Order parameters for Tyr and His aromatic moieties are similar to the order parameters for the corresponding backbone alpha carbon spins reported previously (Palmer, A. G.; Rance, M.; Wright, P. E. J. Am. Chem. Soc. 1991,113, 4371-4380); thus, these aromatic side chains have little additional conformational freedom on picosecond to nanosecond time scales. In contrast, order parameters for Phe 10 indicate that the phenyl ring is more highly restricted than the backbone alpha carbon spin; thus, the order parameters reflect the packing of the Phe 10 side chain in the hydrophobic core of the molecule. Phenomenological spin-spin relaxation rate constants for the two His residues provide evidence that a chemical-exchange process occurs in the zinc-binding site of the peptide. Order parameters for the symmetry axes of the methyl groups are determined from the methyl C-H order parameters assuming tetrahedral geometry. In general, order parameters for the symmetry axes are smaller for residues with longer side chains; however, 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 germinal methyl groups of Val and Leu residues are compared to order parameters obtained previously for the Val beta and Leu gamma methine carbon spins; the observed differences may reflect steric constraints on motions of the methyl and methine groups. The symmetry axis order parameter for Ala 15 is larger than unity and indicates that relaxation is mediated by motions with effective correlation times in excess of 100 ps. Analysis using specific models for internal motions of methyl groups suggests that the motion of this methyl group cannot be described simply by restricted rotational diffusion or three-site jump models. Order parameters measured by NMR and fluorescence spectroscopies for the tyrosyl ring in the zinc complex of the peptide are in good agreement; consequently, order parameters appear to be accurately determined by both experimental techniques. The overall rotational correlation time measured by fluorescence spectroscopy is 19% smaller than that measured by NMR spectroscopy. Fluorescence depolarization is faster in the absence of zinc and probably is dominated by large amplitude local motions of the unfolded peptide.