Slow Exchange in the Chromophore of a Green Fluorescent Protein Variant

Abstract

Green fluorescent protein and its mutants have become valuable tools in molecular biology. They also provide systems rich in photophysical and photochemical phenomena of which an understanding is important for the development of new and optimized variants of GFP. Surprisingly, not a single NMR study has been reported on GFPs until now, possibly because of their high tendency to aggregate. Here, we report the (19)F nuclear magnetic resonance (NMR) studies on mutants of the green fluorescent protein (GFP) and cyan fluorescent protein (CFP) labeled with fluorinated tryptophans that enabled the detection of slow molecular motions in these proteins. The concerted use of dynamic NMR and (19)F relaxation measurements, supported by temperature, concentration- and folding-dependent experiments provides direct evidence for the existence of a slow exchange process between two different conformational states of CFP. (19)F NMR relaxation and line shape analysis indicate that the time scale of exchange between these states is in the range of 1.2-1.4 ms. Thermodynamic analysis revealed a difference in enthalpy (Delta)H(0) = (18.2 +/- 3.8) kJ/mol and entropy T(Delta)S(0) = (19.6 +/- 1.2) kJ/mol at T = 303 K for the two states involved in the exchange process, indicating an entropy-enthalpy compensation. The free energy of activation was estimated to be approximately 60 kJ/mol. Exchange between two conformations, either of the chromophore itself or more likely of the closely related histidine 148, is suggested to be the structural process underlying the conformational mobility of GFPs. The possibility to generate a series of single-atom exchanges ("atomic mutations") like H --> F in this study offers a useful approach for characterizing and quantifying dynamic processes in proteins by NMR.

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

“…For example, the dynamics and spectroscopic characteristics upon fluorotryptophan ((4-F)Trp, (5-F)Trp or (6-F)Trp) incorporation into EGFP and ECFP (enhanced cyan fluorescent protein) have been studied. 109,138 Whereas EGFP contains only a single Trp residue in position 57, ECFP, in addition, harbors Trp66 as a part of the chromophore. Interestingly, in 19 F NMR analyses of the fluorinated EGFP and ECFP congeners, two and four signals were detectable, respectively, reflecting two states of each fluorinated Trp residue.…”

“…For example, Tyr145 and His148 show an increased flexibility indicating some conformational freedom, and they are located in the vicinity of Trp66. 109,141 To date, the related two conformational states of the proteins could only be detected in solution ( 19 F NMR) but not in crystal structures. 138 As the fluorescence properties of these proteins arise mostly from the chromophore, it was expected that the incorporation of fluorotryptophans into EGFP will have no significant effect and indeed there is no difference in the spectral profiles of the congeners and the parent protein.…”

The dual-basin landscape in GFP folding

“…Our coarse-grained model captures some structural aspects of the chromophore that are important in describing folding through a noncanonical kink in the central ␣-helix. Recent work on GFP-family chromophore isomerization using crystallography and molecular dynamics has revealed chromophore isomerization in fluorescent proteins (19)(20)(21) and major differences in the interior hydrogen-bond networks between chromophore isomerizations (22)(23)(24).…”

High-precision FLIM–FRET in fixed and living cells reveals heterogeneity in a simple CFP–YFP fusion protein

“…The origin of these two lifetime components has been attributed to the presence of two different conformations of the chromophore in CFP due to interactions with nearby amino acids [28]. There is evidence from NMR spectroscopy that these two conformations interconvert on a millisecond timescale [29], which is a much slower timescale than the fluorescence decay. Each conformer will, therefore, exist as a distinct emitting species.…”