Fluorine nuclear magnetic resonance analysis of the ligand binding properties of two homologous rat cellular retinol-binding proteins expressed in Escherichia coli.
Comparative 19F NMR studies were performed on rat cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBPII) to better understand their role in intracellular retinol metabolism within the polarized absorptive epithelial cells (enterocytes) of the intestine. Efficient incorporation of 6-fluorotryptophan (6-FTrp) into these homologous proteins was achieved by growing a tryptophan auxotroph of Escherichia coli, harboring prokaryotic expression vectors with either a full-length rat CRBPII or CRBP cDNA on defined medium supplemented with the analog. It is possible to easily distinguish resonances corresponding to 6-FTrp-apoCRBP, 6-FTrp-CRBP-retinol (or retinal), 6-FTrp-apoCRBPII, and 6-FTrp-CRBPII-retinol (or retinal). We were thus able to use 19F NMR spectroscopy to monitor transfer of all-trans-retinol and all-trans-retinal between CRBPII and CRBP in vitro. Retinol complexed to CRBPII is readily transferred to CRBP, whereas retinol complexed to CRBP is not readily transferred to CRBPII. We estimated that the Kd for CRBP-retinol is approximately 100-fold less than the Kd for CRBPII-retinol. Transfer of all-trans-retinal occurs readily from CRBPII to CRBP and from CRBP to CRBPII. Results from competitive binding studies with retinol and retinal indicated that there is a much larger difference between the affinities of CRBP for retinol and retinal than between the affinities of CRBPII for these two ligands. However, the differences in binding specificities reflect differences in how the two proteins interact with retinol, rather than with retinal. 19F NMR analysis of recombinant isotopically labeled proteins represents a sensitive new and useful method for monitoring retinoid flux between the CRBPs in vitro.
Li
E
E
Department of Medicine, Washington University, St. Louis, Missouri 63110.
Qian
S J
SJ
Winter
N S
NS
d'Avignon
A
A
Levin
M S
MS
Gordon
J I
JI
D000818:Animals; D004926:Escherichia coli / Q000235:genetics*; D005461:Fluorine; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D051381:Rats; D012177:Retinol-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D054840:Retinol-Binding Proteins, Cellular; D013050:Spectrometry, Fluorescence
D008024:Ligands; D012177:Retinol-Binding Proteins; D054840:Retinol-Binding Proteins, Cellular; D005461:Fluorine