Characterization of two fluorescent tryptophans in recombinant human granulocyte-colony stimulating factor: comparison of native sequence protein and tryptophan-deficient mutants.
In order to probe the role of the individual tryptophans of granulocyte-colony stimulating factor (G-CSF) in pH and guanidine HCl-induced fluorescence changes, site-directed mutagenesis was used to generate mutants replacing Trp118,Trp58, or both with phenylalanine. Neither Trp to Phe mutation affected the folding or activity of the recombinant G-CSF, and the material expressed in yeast behaved identically to that expressed in Escherichia coli. All of the G-CSF species responded to pH and guanidine HCl in qualitatively the same manner. Trp58 has a fluorescence maximum at 350 nm and is quenched to a greater extent by the addition of guanidine HCl, indicating that it is fully solvent-exposed. Trp118 has a fluorescence maximum at 344 nm, and is less solvent-accessible than Trp58. The analog in which both tryptophans have been replaced with phenylalanine shows only tyrosine fluorescence, with a peak at 304 nm which decreases with increasing pH. The intensity of the tyrosine fluorescence in this analog is much greater than that of the native sequence protein or single tryptophan mutants, indicating that energy transfer is taking place from tyrosine to tryptophan in these molecules. Below neutral pH the tyrosine fluorescence is much greater in the [Phe58]G-CSF than in the [Phe118]G-CSF, indicating that Trp58 might be a more efficient recipient of energy transfer from the tyrosine(s).
Kolvenbach
C G
CG
Amgen, Inc., Amgen Center, Thousand Oaks, California 91320.
Elliott
S
S
Sachdev
R
R
Arakawa
T
T
Narhi
L O
LO
D003001:Cloning, Molecular; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000235:genetics; D019791:Guanidine; D006146:Guanidines; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
D006146:Guanidines; D016179:Granulocyte Colony-Stimulating Factor; D014364:Tryptophan; D019791:Guanidine