F-19 NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPIC STUDY OF FLUOROPHENYLALANINE-LABELED AND FLUOROTRYPTOPHAN-LABELED AVIAN EGG-WHITE LYSOZYMES

We report the 470-MHz (11.7 T) F-19 solution nuclear magnetic resonanc e (NMR) spectra of 2-, 3-, and 4-fluorophenylalanine incorporated into the egg white lysozymes (EC 3.2.1.17) of chicken, pheasant, and duck, as well as spectra of 4-fluorotryptophan incorporated into chicken, C alifornia valley quail, and Bob White quail and 5- and 6-fluorotryptop han-labeled chicken lysozyme. The F-19 solution NMR spectrum of [4-F]P he hen egg white lysozyme (HEWL) consists of three sharp resonances, w hich span a total chemical shift range of 4.8 ppm (at p(2)H = 6.1). Fo r [3-F]Phe HEWL, the chemical shift range is much smaller, 1.0 ppm (at p(2)H = 5.9), due presumably to the occurrence of fast phenyl ring fl ips about the C-beta-C-gamma bond axis. For [2-F]Phe HEWL, six resonan ces are observed, spanning a chemical shift range of 7.4 ppm (at p(2)H = 5.8), due to slow C-beta-C-gamma ring flips, i.e., both ring-flip i somers appear to be ''frozen in'' because of steric hindrance. Rotatio n of the [2-F]Phe residues remains slow up to 55 degrees C (p(2)H = 4. 7). With the [F]Trp-labeled proteins, we find a maximal 14.6-ppm shiel ding 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 e xposure 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 sm all chemical shift changes (<1.4 ppm) over the pH range of similar to 1.2-7.8. In addition, spectra of highly acetylated [4-F]Phe and [4-F]T rp 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 i s 3.71 Angstrom from the N-terminal lysine) becomes shielded by simila r to 1.5 ppm on acetylation. About 1-1.5-ppm shielding changes were al so 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 differenc es (e.g., Val<->Ile, Ser<->Thr) rather than to surface charge field mo difications (Gln-->His). These results suggest that surface charge fie lds make only a small contribution to F-19 shielding. Preliminary assi gnments of [4-F]Trp HEWL expressed in Saccharomyces cerevisiae have be en made by using W62Y and W63Y mutants, and H-2 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. Final ly, we investigated the effects of inhibitor binding to [4-F]Trp HEWL. We find fast, intermediate, and slow chemical exchange behavior, resp ectively, on binding N-acetyl-D-glucosamine, N,N'-diacetylchitobiose, and N,N'N''-triacetylchiototriose ((NAG)(3)) inhibitors. There are mod est (similar to 2 ppm) shielding changes for two resonances, tentative ly assigned to Trp-63 and Trp-108, with the 16.8-ppm F-19 chemical shi ft range for [4-F]Trp HEWL/(NAC)(3) being the largest observed so far in proteins. Overall, our results indicate that F-19-labeled amino aci ds can be readily incorporated (within a few days) into avian lysozyme s, that spectra can begin to be assigned by means of interspecies comp arisons and site-directed mutagenesis, that ortho fluorine substitutio n presents a large steric hindrance to phenyl ring rotation, and that surface charge fields play only a small role in F-19 shielding, while (neutral) inhibitor binding or small amino acid side-chain changes app ear to cause larger shielding effects than do surface charge field mod ifications.