Fluorine-19 nuclear magnetic resonance spectroscopic study of fluorophenylalanine- and fluorotryptophan-labeled avian egg white lysozymes.
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 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 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 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 p2H = 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 (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 approximately 1.2-7.8.(ABSTRACT TRUNCATED AT 250 WORDS)
Lian
C
C
Department of Chemistry, University of Illinois at Urbana-Champaign 61801.
Le
H
H
Montez
B
B
Patterson
J
J
Harrell
S
S
Laws
D
D
Matsumura
I
I
Pearson
J
J
Oldfield
E
E
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D005260:Female; D005461:Fluorine; D019791:Guanidine; D006146:Guanidines; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D013058:Mass Spectrometry; D009113:Muramidase / Q000096:biosynthesis / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000032:analysis; D011487:Protein Conformation; D011489:Protein Denaturation; D011784:Quail; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D014364:Tryptophan / Q000031:analogs & derivatives / Q000032:analysis; D010135:p-Fluorophenylalanine / Q000032:analysis
D006146:Guanidines; D011994:Recombinant Proteins; C039390:4-fluorotryptophan; D005461:Fluorine; C027217:2-fluorophenylalanine; C027218:3-fluorophenylalanine; D010649:Phenylalanine; D010135:p-Fluorophenylalanine; D014364:Tryptophan; D009113:Muramidase; D019791:Guanidine