Summary
A high-resolution, solution-state NMR method for characterizing and comparing the interactions between carboxyl 13C-enriched fatty acids (FA) and individual binding sites on proteins has been developed. The utility of this method results from the high degree of resolution of carboxyl from other carbon resonances and the high sensitivity of FA carboxyl chemical shifts to intermolecular environmental factors such as degree of hydrogen-bonding or hydration, degree of ionization (pH), and proximity to positively-charged or aromatic side-chain moieties in proteins. Information can be obtained regarding binding heterogeneity (structural as well as thermodynamic), binding stoichiometries, relative binding affinities, the ionization behavior of bound FA and protein side-chain moieties, the physical and ionization states of unbound FA, and the exchange rates of FA between protein binding sites and between protein and non-protein acceptors of FA, such as model membranes.
Cytosolic fatty acid binding proteins represent an excellent model system for studying and comparing fatty acid-protein interactions. Prokaryotic expression vectors have been used to direct efficient synthesis of several mammalian intestinal FABPs in E. coli. This has enabled us to isolate gram-quantities of purified FABPs, to introduce NMR-observable isotopes, and to generate FABP mutants.
The intestine is the only tissue known to contain abundant quantities of more than one FABP homologue in a single cell type. It is likely that these homologous FABPs serve distinct functional roles in intestinal lipid transport. This paper presents comparative 13C NMR results for FA interactions with FABP homologues from intestine, and the functional implications of these analyses are discussed.
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Abbreviations
- FA:
-
Fatty Acid(s)
- FABP:
-
Fatty Acid Binding Protein(s)
- I-FABPc :
-
Cytosolic rat intestinal Fatty Acid Binding Protein
- L-FABPc :
-
Cytosolic rat liver Fatty Acid Binding Protein
- CD:
-
Circular Dichroic spectroscopy
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Established Investigator of the American Heart Association
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Cistola, D.P., Sacchettini, J.C. & Gordon, J.I. 13C NMR studies of fatty acid-protein interactions: comparison of homologous fatty acid-binding proteins produced in the intestinal epithelium. Mol Cell Biochem 98, 101–110 (1990). https://doi.org/10.1007/BF00231373
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DOI: https://doi.org/10.1007/BF00231373