Backbone dynamics of green fluorescent protein and the effect of histidine 148 substitution.
Green fluorescent protein (GFP) and its mutants have become valuable tools in molecular biology. GFP has been regarded as a very stable and rigid protein with the beta-barrel shielding the chromophore from the solvent. Here, we report the 15N nuclear magnetic resonance (NMR) studies on the green fluorescent protein (GFPuv) and its mutant His148Gly. 15N NMR relaxation studies of GFPuv show that most of the beta-barrel of GFP is rigid on the picosecond to nanosecond time scale. For several regions, including the first alpha-helix and beta-sheets 3, 7, 8, and 10, increased hydrogen-deuterium exchange rates suggest a substantial conformational flexibility on the microsecond to millisecond time scales. Mutation of residue 148 located in beta-sheet 7 is known to have a strong impact on the fluorescence properties of GFPs. UV absorption and fluorescence spectra in combination with 1H-15N NMR spectra indicate that the His148Gly mutation not only reduces the absorption of the anionic chromophore state but also affects the conformational stability, leading to the appearance of doubled backbone amide resonances for a number of residues. This suggests the presence of two conformations in slow exchange on the NMR time scale in this mutant.
Seifert
Markus H J
MH
Max-Planck-Institute for Biochemistry, 82152 Martinsried, Germany, and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada. markus.seifert@4sc.com
Georgescu
Julia
J
Ksiazek
Dorota
D
Smialowski
Pawel
P
Rehm
Till
T
Steipe
Boris
B
Holak
Tad A
TA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D018360:Crystallography, X-Ray; D003903:Deuterium; D005998:Glycine / Q000235:genetics; D049452:Green Fluorescent Proteins; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D010449:Peptide Mapping; D017354:Point Mutation; D014176:Protein Biosynthesis; D011487:Protein Conformation; D017433:Protein Structure, Secondary / Q000235:genetics; D011522:Protons; D037401:Scyphozoa; D016415:Sequence Alignment; D013816:Thermodynamics
D008164:Luminescent Proteins; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D011522:Protons; D049452:Green Fluorescent Proteins; D006639:Histidine; D003903:Deuterium; D005998:Glycine