Fluorine-19 Nuclear Magnetic Resonance Spectroscopic Study of Fluorophenylalanine- and Fluorotryptophan-Labeled Avian Egg White Lysozymes

Chenyang Lian, Hongbiao Le, Bernard Montez, Jessica Patterson, Shannon Harrell, David Laws, John Pearson, Eric Oldfield, Ichiro Matsumura

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We report the 470-MHz (11.7 T) 19F solution nuclear magnetic resonance (NMR) spectra of 2-, 3-, and 4-fluorophenylalanine incorporated into the egg white lysozymes (EC of chicken, pheasant, and duck, as well as spectra of 4-fluorotryptophan incorporated into chicken, California valley quail, and Bob White quail and 5- and 6-fluorotryptophan-labeled chicken lysozyme. The 19F solution NMR spectrum of [4-F]Phe hen egg white lysozyme (HEWL) consists of three sharp resonances, which span a total chemical shift range of 4.8 ppm (at p2H = 6.1). For [3-F]Phe HEWL, the chemical shift range is much smaller, 1.0 ppm (at p2H = 5.9), due presumably to the occurrence of fast phenyl ring flips about the Cβ-Cγ bond axis. For [2-F]Phe HEWL, six resonances are observed, spanning a chemical shift range of 7.4 ppm (at p2H = 5.8), due to slow Cβ-Cγ ring flips, i.e., both ring-flip isomers appear to be “frozen in” because of steric hindrance. Rotation of the [2-F]Phe residues remains slow up to 55 °C (p2H = 4.7). With the [F]Trp-labeled proteins, we find a maximal 14.6-ppm shielding range for [4-F]Trp HEWL but only a 2.8- and 2.4-ppm range for [5- and 6-F]Trp HEWL, respectively, due presumably to increased solvent exposure in the latter cases. Guanidinium chloride denaturation causes loss of essentially all chemical shift nonequivalence, as does thermal denaturation. Spectra recorded as a function of pH show relatively small chemical shift changes (< 1.4 ppm) over the pH range of ∼ 1.2–7.8. In addition, spectra of highly acetylated [4-F]Phe and [4-F]Trp HEWLs, in which most lysine side chains are converted to (neutral) acetamides (as determined by electrospray ionization mass spectrometry) also show only minor chemical shift changes, although Phe-3 (which is 3.71 Å from the N-terminal lysine) becomes shielded by ∼ 1.5 ppm on acetylation. About 1–1.5-ppm shielding changes were also seen among the [4-F]Trp lysozymes of hen, California valley quail, and Bob White quail and appear to be due to minor side-chain differences (e.g., Val-Ile, Ser↔Thr) rather than to surface charge field modifications (Gin→His). These results suggest that surface charge fields make only a small contribution to 19F shielding. Preliminary assignments of [4-F]Trp HEWL expressed in Saccharomyces cerevisiae have been made by using W62Y and W63Y mutants, and 2H solvent-induced shifts were consistent with these assignments. Iodine and N-bromosuccinimide oxidation and TEMPO acetamide and Gd3+ binding cause line-broadening, which yields tentative assignments for some of the other peaks. Finally, we investigated the effects of inhibitor binding to [4-F]Trp HEWL. We find fast, intermediate, and slow chemical exchange behavior, respectively, on binding A-acetyl-D-glucosamine, N,N-diacetylchitobiose, and N,N′,N″-triacetylchiototriose ((NAG)3) inhibitors. There are modest (∼2 ppm) shielding changes for two resonances, tentatively assigned to Trp-63 and Trp-108, with the 16.8-ppm 19F chemical shift range for [4-F]Trp HEWL/(NAG)3 being the largest observed so far in proteins. Overall, our results indicate that 19F-labeled amino acids can be readily incorporated (within a few days) into avian lysozymes, that spectra can begin to be assigned by means of interspecies comparisons and site-directed mutagenesis, that ortho fluorine substitution presents a large steric hindrance to phenyl ring rotation, and that surface charge fields play only a small role in 19F shielding, while (neutral) inhibitor binding or small amino acid side-chain changes appear to cause larger shielding effects than do surface charge field modifications.

Original languageEnglish (US)
Pages (from-to)5238-5245
Number of pages8
Issue number17
StatePublished - May 1 1994

ASJC Scopus subject areas

  • Biochemistry


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