J. Biol. Chem., Vol. 264, Issue 29, 17041-17048, 10, 1989
Nuclear magnetic resonance studies of 6-fluorotryptophan-substituted rat cellular retinol-binding protein II produced in Escherichia coli. Analysis of the apoprotein and the holoprotein containing bound all- trans-retinol and all-trans-retinal
E Li, SJ Quian, L Nader, NC Yang, A d'Avignon, JC Sacchettini and JI Gordon
Department of Medicine, Washington University, St. Louis, Missouri 63110.
Rat cellular retinol-binding protein II (CRBP II) is a 15.6-kDa intestinal
protein which binds all-trans-retinol and all-trans-retinal but not
all-trans-retinoic acid. We have previously analyzed the interaction of
Escherichia coli-derived rat apoCRBP II with several retinoids using
fluorescence spectroscopic techniques. Interpretation of these experiments
is complicated, because the protein has 4 tryptophan residues. To further
investigate ligand-protein interactions, we have utilized 19F nuclear
magnetic resonance (NMR) spectroscopy of CRBP II labeled at its 4
tryptophan residues with 6- fluorotryptophan. Efficient incorporation of
6-fluorotryptophan (93%) was achieved by growing a tryptophan auxotroph of
E. coli harboring a prokaryotic expression vector with a full-length rat
CRBP II cDNA on defined medium supplemented with the analog. Comparison of
the 19F NMR spectra of 6-fluorotryptophan-substituted CRBP II with and
without bound all-trans-retinol revealed that resonances corresponding to 2
tryptophan residues (designated WA and WB) undergo large downfield changes
in chemical shifts (2.0 and 0.5 ppm, respectively) associated with ligand
binding. In contrast, 19F resonances corresponding to two other tryptophan
residues (WC and WD) undergo only minor perturbations in chemical shifts.
The 19F NMR spectra of 6-fluorotryptophan- substituted CRBP II complexed
with all-trans-retinal and all-trans- retinol were very similar, suggesting
that the interactions of these two ligands with the protein are similar.
Molecular model building, based on the crystalline structures of two
homologous proteins was used to predict the positions of the 4 tryptophan
residues of CRBP II and to make tentative resonance assignments. The fact
that ligand binding produced residue-specific changes in the chemical
shifts of resonances in CRBP II suggests that NMR analysis of isotopically
labeled retinoid- binding proteins expressed in E. coli will provide an
alternate, albeit it complementary, approach to fluorescence spectroscopy
for examining the structural consequences of their association with ligand.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?