¹⁹F NMR Studies of the Native and Denatured States of Green Fluorescent Protein

Abstract

Biosynthetic preparation and (19)F NMR experiments on uniformly 3-fluorotyrosine-labeled green fluorescent protein (GFP) are described. The (19)F 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 (19)F photo-CIDNP (chemically induced dynamic nuclear polarization) experiments, yielded a full (19)F NMR assignment. Two (19)F-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 (19)F 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 (19)F-labeled tyrosine side chains and the (19)F-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.

The extremely slow‐exchanging core and acid‐denatured state of green fluorescent protein

“…Such residual structure is likely to reduce the initial conformational search the polypeptide chain has to undertake during the first steps of folding and this may lead to faster and more efficient folding. For GFP, this is supported by results from folding studies which have shown that the refolding efficiency and rate from the acid denatured state (Fukuda et al, 2000;Enoki et al, 2004), which we and others have shown contains significant residual structure, is considerably higher than from the chemically denatured state produced by high concentrations of GdmCl which appears to have little or no residual structure (Khan et al, 2006).…”

“…From studies of GFP, it is known that there is considerable residual structure under acid-denaturing conditions as shown by far-UV CD (Enoki et al, 2004), small-angle x-ray scattering (Enoki et al, 2006) and 19 F-NMR photo-CINDP (Khan et al, 2006) experiments, however, it was not know which regions of the protein were involved in the residual structure. Here, we have identified the residues associated with this residual structure and shown that they locate to ␤-strands 1 and 3.…”

“…Studies on the properties of denatured states have therefore become an important area in the study of protein folding pathways. From far-ultra violet (UV) circular dichroism (CD), it is clear that there is residual secondary structure in the acid-denatured state of GFP (Enoki et al, 2004) results supported by small-angle x-ray scattering studies (Enoki et al, 2006) and 19 F-NMR/photo-CIDNP experiments (Khan et al, 2006). Other than the fact that residual structure is present in the denatured state of GFP at low pH, little is known about which regions of the protein are involved.…”

Organic fluorine as a polypeptide building element: in vivo expression of fluorinated peptides, proteins and proteomes

“…This could also be confirmed by the NMR studies of Khan and co-workers. 108 They used a truncated mutant of GFPuv and mutated each Tyr residue one after the other to Phe in order to assign the NMR signals. From their experiments they concluded that ring-flipping must be a slow process and is in a time frame of 160 s −1 for (3-F)Tyr143 and 960 s −1 for (3-F)Tyr92.…”

A pre-existing hydrophobic collapse in the unfolded state of an ultrafast folding protein

“…Applications to proteins that fold more slowly than ~100 ms will require either more dramatic hyperpolarizations 22,29 or slower nuclear spin-lattice relaxation 30 . We therefore anticipate that this approach will provide valuable information on the structures of denatured states of many proteins, particularly those that refold rapidly.…”