Liver fatty acid binding protein : relating structure to function

Abstract

Liver Fatty Acid Binding Protein (LFABP) is a member of a family of small (14 kDa) intracellular proteins that reversibly bind fatty acids. This binding greatly enhances their aqueous solubility and thus facilitates their transport.

In this investigation the involvement of cationic α-helical residues (K20, K31 and K33) in ligand binding and residues contributing to the positive surface charge of the protein (K36, K47 and K57) in ligand binding and interaction with phospholipid vesicles has been studied by using charge reversal mutagenesis to convert these residues to glutamate.  It was found that the three residues that made the largest contribution in terms of ligand binding and in regards to the protein’s interaction with phospholipid vesicles were K31, K36 and K57.  These residues are positioned around the hypothesized portal region of LFABP.

The tryptophan insertion mutants of LFABP, L28W, Y54W and M74W, were produced.  Ligand binding studies highlighted L28W as a mutant that demonstrated dramatically enhanced fluorescence on fatty acid binding.  Detailed studies on the mutant made it possible to show co-operative binding of oleic acid to LFABP and to carry out direct competition assays between oleic acid and oleoyl CoA.  These competition assays found that these two ligands have similar affinities in terms of binding to LFABP, a result that is important in assigning the role of LFABP within the cell.  A further tryptophan mutant Y7W, has been used to investigate the conformational change at the protein’s N-terminal region, complementing previous studies with the mutant F3W.

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