Probing local environments of tryptophan residues in proteins: Comparison of F-19 nuclear magnetic resonance results with the intrinsic fluorescence of soluble human tissue factor

F-19 nuclear magnetic resonance (F-19 NMR) of 5-fluorotryptophan (5F-Trp) a nd tryptophan (Trp) fluorescence both provide information about local envir onment and solvent exposure of Trp residues. To compare the information pro vided by these spectroscopies, the four Trp residues in recombinant soluble human tissue factor (sTF) were replaced with 5F-Trp, F-19 NMR assignments for the SF-Trp residues (14, 25, 45, and 158) were based on comparison of t he wild-type protein spectrum with the spectra of three single Trp-to-Phe r eplacement mutants, Previously we showed from fluorescence and absorption d ifference spectra of mutant versus wild-type sTF that the side chains of Tr p14 and Trp25 are buried, whereas those of Trp45 and Trp158 are partially e xposed to bulk solvent (Hasselbacher et al,, Biophys J 1995;69:20-29), F-19 NMR paramagnetic broadening and solvent-induced isotope-shift experiments show that position 5 of the indole ring of 5F-Trp158 is exposed, whereas th at of 5F-Trp45 is essentially inaccessible. Although BF-Trp incorporation h ad no discernable effect on the procoagulant cofactor activity of either th e wild-type or mutant proteins, F-19 NMR chemical shifts showed that the si ngle-Trp mutations are accompanied by subtle changes in the local environme nts of 5F-Trp residues residing in the same structural domain. Proteins 199 9;37:709-716, (C) 1999 Wiley-Liss, Inc.