| Autor: |
Kolvenbach, Carl G., Elliott, Steven, Sachdev, Raj, Arakawa, Tsutomu, Narhi, Linda O. |
| Zdroj: |
Journal of Protein Chemistry; April 1993, Vol. 12 Issue: 2 p229-236, 8p |
| Abstrakt: |
In order to probe the role of the individual tryptophans of granulocyte-colony stimulating factor (G-CSF) inpH 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 inEscherichia coli. All of the G-CSF species responded topH and guanidine HCl in qualitatively the same manner. Trp58has 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. Trp118has 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 increasingpH. 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 neutralpH the tyrosine fluorescence is much greater in the [Phe58]G-CSF than in the [Phe118]G-CSF, indicating that Trp58might be a more efficient recipient of energy transfer from the tyrosine(s). |
| Databáze: |
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