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

19F NMR studies of the native and denatured states of green fluorescent protein
19F NMR studies of the native and denatured states of green fluorescent protein
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.
0002-7863
10729-10737
Khan, Farid
bd001c47-9aab-4b1b-9528-e7d343f83119
Kuprov, Ilya
bb07f28a-5038-4524-8146-e3fc8344c065
Craggs, Timothy D.
7fad3c33-3a94-4745-a17e-3c600303785d
Hore, P.J.
cad4561e-9571-4b49-b633-1c0bb470d144
Jackson, Sophie E.
7be9b16e-329b-49fb-ac75-938a297ee4b8
Khan, Farid
bd001c47-9aab-4b1b-9528-e7d343f83119
Kuprov, Ilya
bb07f28a-5038-4524-8146-e3fc8344c065
Craggs, Timothy D.
7fad3c33-3a94-4745-a17e-3c600303785d
Hore, P.J.
cad4561e-9571-4b49-b633-1c0bb470d144
Jackson, Sophie E.
7be9b16e-329b-49fb-ac75-938a297ee4b8

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).

Record type: Article

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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More information

e-pub ahead of print date: 29 July 2006
Published date: 2006
Organisations: Computational Systems Chemistry

Identifiers

Local EPrints ID: 347391
URI: http://eprints.soton.ac.uk/id/eprint/347391
ISSN: 0002-7863
PURE UUID: 052041e5-8589-4004-9246-96d71c120f05
ORCID for Ilya Kuprov: ORCID iD orcid.org/0000-0003-0430-2682

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Date deposited: 27 Feb 2013 12:15
Last modified: 15 Mar 2024 03:43

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Contributors

Author: Farid Khan
Author: Ilya Kuprov ORCID iD
Author: Timothy D. Craggs
Author: P.J. Hore
Author: Sophie E. Jackson

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