19F NMR studies of the native and denatured states of green fluorescent protein

Khan, Farid, Kuprov, Ilya, Craggs, Timothy D., Hore, P.J. and Jackson, Sophie E. (2006) 19F NMR studies of the native and denatured states of green fluorescent protein. Journal of the American Chemical Society, 128 (33), 10729-10737. (doi:10.1021/ja060618u).

Abstract

Biosynthetic preparation and 19F NMR experiments on uniformly 3-fluorotyrosine-labeled green fluorescent protein (GFP) are described. The 19F NMR signals of all 10 fluorotyrosines are resolved in the protein spectrum with signals spread over 10 ppm. Each tyrosine in GFP was mutated in turn to phenylalanine. The spectra of the Tyr ? Phe mutants, in conjunction with relaxation data and results from 19F photo-CIDNP (chemically induced dynamic nuclear polarization) experiments, yielded a full 19F NMR assignment. Two 19F-Tyr residues (Y92 and Y143) were found to yield pairs of signals originating from ring-flip conformers; these two residues must therefore be immobilized in the native structure and have 19F nuclei in two magnetically distinct positions depending on the orientation of the aromatic ring. Photo-CIDNP experiments were undertaken to probe further the structure of the native and denatured states. The observed NMR signal enhancements were found to be consistent with calculations of the HOMO (highest occupied molecular orbital) accessibilities of the tyrosine residues. The photo-CIDNP spectrum of native GFP shows four peaks corresponding to the four tyrosine residues that have solvent-exposed HOMOs. In contrast, the photo-CIDNP spectra of various denatured states of GFP show only two peaks corresponding to the 19F-labeled tyrosine side chains and the 19F-labeled Y66 of the chromophore. These data suggest that the pH-denatured and GdnDCl-denatured states are similar in terms of the chemical environments of the tyrosine residues. Further analysis of the sign and amplitude of the photo-CIDNP effect, however, provided strong evidence that the denatured state at pH 2.9 has significantly different properties and appears to be heterogeneous, containing subensembles with significantly different rotational correlation times.

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Contributors

Author: Ilya Kuprov

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