Probing local environments of tryptophan residues in proteins: Comparison of 19F nuclear magnetic resonance results with the intrinsic fluorescence of soluble human tissue factor
✍ Scribed by Jennifer Zemsky; Elena Rusinova; Yale Nemerson; Linda A. Luck; J.B. Alexander Ross
- Publisher
- John Wiley and Sons
- Year
- 1999
- Tongue
- English
- Weight
- 165 KB
- Volume
- 37
- Category
- Article
- ISSN
- 0887-3585
No coin nor oath required. For personal study only.
✦ Synopsis
19 F nuclear magnetic resonance ( 19 F NMR) of 5-fluorotryptophan (5F-Trp) and tryptophan (Trp) fluorescence both provide information about local environment and solvent exposure of Trp residues. To compare the information provided by these spectroscopies, the four Trp residues in recombinant soluble human tissue factor (sTF) were replaced with 5F-Trp. 19 F NMR assignments for the 5F-Trp residues (14, 25, 45, and 158) were based on comparison of the wild-type protein spectrum with the spectra of three single Trp-to-Phe replacement mutants. Previously we showed from fluorescence and absorption difference spectra of mutant versus wild-type sTF that the side chains of Trp14 and Trp25 are buried, whereas those of Trp45 and Trp158 are partially exposed to bulk solvent (Hasselbacher et al., Biophys J 1995;69:20-29). 19 F NMR paramagnetic broadening and solvent-induced isotope-shift experiments show that position 5 of the indole ring of 5F-Trp158 is exposed, whereas that of 5F-Trp45 is essentially inaccessible. Although 5F-Trp incorporation had no discernable effect on the procoagulant cofactor activity of either the wild-type or mutant proteins, 19 F NMR chemical shifts showed that the single-Trp mutations are accompanied by subtle changes in the local environments of 5F-Trp residues residing in the same structural domain. Pro-