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Titolo:
F-19 NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPIC STUDY OF FLUOROPHENYLALANINE-LABELED AND FLUOROTRYPTOPHAN-LABELED AVIAN EGG-WHITE LYSOZYMES
Autore:
LIAN CY; LE HB; MONTEZ B; PATTERSON J; HARRELL S; LAWS D; MATSUMURA I; PEARSON J; OLDFIELD E;
Indirizzi:
UNIV ILLINOIS,DEPT CHEM,505 S MATHEWS AVE URBANA IL 61801 UNIV ILLINOIS,DEPT CHEM URBANA IL 61801 UNIV CALIF BERKELEY,DEPT MOLEC & CELL BIOL BERKELEY CA 94720
Titolo Testata:
Biochemistry
fascicolo: 17, volume: 33, anno: 1994,
pagine: 5238 - 5245
SICI:
0006-2960(1994)33:17<5238:FNSSOF>2.0.ZU;2-0
Fonte:
ISI
Lingua:
ENG
Soggetto:
GALACTOSE CHEMOSENSORY RECEPTOR; AMINO-ACID-SEQUENCE; FLUOROTYROSINE ALKALINE-PHOSPHATASE; INDUCED CONFORMATIONAL-CHANGES; CHEMICAL-SHIFT ANISOTROPY; D-LACTATE DEHYDROGENASE; F-19 NMR; ESCHERICHIA-COLI; DIHYDROFOLATE-REDUCTASE; STRUCTURAL-CHANGE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
55
Recensione:
Indirizzi per estratti:
Citazione:
C.Y. Lian et al., "F-19 NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPIC STUDY OF FLUOROPHENYLALANINE-LABELED AND FLUOROTRYPTOPHAN-LABELED AVIAN EGG-WHITE LYSOZYMES", Biochemistry, 33(17), 1994, pp. 5238-5245

Abstract

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 causesloss 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 to1.2-7.8. 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 farin 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 thatsurface 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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Documento generato il 03/12/20 alle ore 14:51:45