Environment of tryptophan 57 in porcine fructose-1,6-bisphosphatase studied by time-resolved fluorescence and site-directed mutagenesis.
The environment of Trp57, introduced by the mutation of a tyrosine in the dynamic loop of porcine liver fructose-1,6-bisphosphatase (FBPase), was examined using time-resolved fluorescence and directed mutation. The Trp57 enzyme was studied previously by X-ray crystallography and steady-state fluorescence, the latter revealing an unexpected redshift in the wavelength of maximum fluorescence emission for the R-state conformer. The redshift was attributed to the negative charge of Asp127 in contact with the indole side chain of Trp57. Time-resolved fluorescence experiments here reveal an indole side chain less solvent exposed and more rigid in the R-state, than in the T-state of the enzyme, consistent with X-ray crystal structures. Replacement of Asp127 with an asparagine causes a 6 nm blueshift in the wavelength of maximum fluorescence emission for the R-state conformer, with little effect on the emission maximum of the T-state enzyme. The data here support the direct correspondence between X-ray crystal structures of FBPase and conformational states of the enzyme in solution, and provide a clear example of the influence of microenvironment on the fluorescence properties of tryptophan.
Wen
J
J
Department of Chemistry,  Iowa State University, Ames, IA, USA.
Nelson
S W
SW
Honzatko
R B
RB
Fromm
H J
HJ
Petrich
J W
JW
D019943:Amino Acid Substitution; D000818:Animals; D001216:Asparagine; D001224:Aspartic Acid; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D006597:Fructose-Bisphosphatase / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013552:Swine; D014364:Tryptophan
D021122:Protein Subunits; D011994:Recombinant Proteins; D001224:Aspartic Acid; D001216:Asparagine; D014364:Tryptophan; D006597:Fructose-Bisphosphatase