Slow exchange in the chromophore of a green fluorescent protein variant.
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.
Seifert
Markus H J
MH
Max-Planck-Institute for Biochemistry, 82152 Martinsried, Munich, Germany.
Ksiazek
Dorota
D
Azim
M Kamran
MK
Smialowski
Pawel
P
Budisa
Nediljko
N
Holak
Tad A
TA
D005461:Fluorine / Q000737:chemistry; D049452:Green Fluorescent Proteins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D005461:Fluorine