ANALYSIS OF A SERIES OF PHENYLALANINE-57 MUTANTS OF THE ADIPOCYTE LIPID-BINDING PROTEIN

The importance of phenylalanine 57, an adipocyte lipid-binding protein (ALBP) portal residue, to ligand affinity and specificity has been in vestigated using a series of ALBP position 57 mutants. In wild-type AL BP, phenylalanine 57 undergoes a side chain rotation upon ligand bindi ng, moving from an inwardly oriented, ligand-exclusive position in apo protein structures to an outwardly oriented position in the holoprotei n. To examine the role of F57 side chain rotation in the apoprotein-ho loprotein transition and in ligand selectivity, ALBP site-specific mut ants F57A, F57G, F57H, and F57W were expressed in Escherichia coli and purified to homogeneity. Mutants were analyzed for binding characteri stics and stability toward chemical denaturation, and energy-minimized models of each mutant were constructed using ape, oleate-, and arachi donate-bound ALBP crystallographic coordinates. The stability of ALBP forms (wtALBP approximate to F57G > F57A, F57W > F57H) was unrelated t o the affinity of ALBP forms (wtALBP approximate to F57W > F57H > F57G > F57A) for various lipids and did not vary between fatty acids. Sinc e ligand selectivity was maintained between wild type and all mutants while ligand affinity was grossly diminished, we conclude that phenyla lanine 57 is critical to the formation of the fatty acid/ALBP complex, but is uninvolved in determination of selectivity over the range of p hysiological ligands tested.