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

被引:43
作者
LIAN, CY
LE, HB
MONTEZ, B
PATTERSON, J
HARRELL, S
LAWS, D
MATSUMURA, I
PEARSON, J
OLDFIELD, E
机构
[1] UNIV ILLINOIS, DEPT CHEM, URBANA, IL 61801 USA
[2] UNIV CALIF BERKELEY, DEPT MOLEC & CELL BIOL, BERKELEY, CA 94720 USA
关键词
D O I
10.1021/bi00183a029
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We report the 470-MHz (11.7 T) F-19 solution nuclear magnetic resonance (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, California valley quail, and Bob White quail and 5- and 6-fluorotryptophan-labeled chicken lysozyme. The F-19 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 p(2)H = 6.1). For [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 flips about the C-beta-C-gamma bond axis. For [2-F]Phe HEWL, six resonances 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 isomers appear to be ''frozen in'' because of steric hindrance. Rotation 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 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 similar to 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 Angstrom from the N-terminal lysine) becomes shielded by similar to 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 (Gln-->His). These results suggest that surface charge fields make only a small contribution to F-19 shielding. Preliminary assignments of [4-F]Trp HEWL expressed in Saccharomyces cerevisiae have been 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. 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 N-acetyl-D-glucosamine, N,N'-diacetylchitobiose, and N,N'N''-triacetylchiototriose ((NAG)(3)) inhibitors. There are modest (similar to 2 ppm) shielding changes for two resonances, tentatively assigned to Trp-63 and Trp-108, with the 16.8-ppm F-19 chemical shift 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 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 F-19 shielding, while (neutral) inhibitor binding or small amino acid side-chain changes appear to cause larger shielding effects than do surface charge field modifications.
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页码:5238 / 5245
页数:8
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