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 investigated using a series of ALBP position 57 mutants. In wild-type ALBP, phenylalanine 57 undergoes a side chain rotation upon ligand binding, moving from an inwardly oriented, ligand-exclusive position in apoprotein structures to an outwardly oriented position in the holoprotein. To examine the role of F57 side chain rotation in the apoprotein-holoprotein transition and in ligand selectivity, ALBP site-specific mutants F57A, F57G, F57H, and F57W were expressed in Escherichia coli and purified to homogeneity. Mutants were analyzed for binding characteristics and stability toward chemical denaturation, and energy-minimized models of each mutant were constructed using apo, oleate-, and arachidonate-bound ALBP crystallographic coordinates. The stability of ALBP forms (wtALBP approximately F57G > F57A > F57W > F57H) was unrelated to the affinity of ALBP forms (wtALBP approximately F57W > F57H > F57G > F57A) for various lipids and did not vary between fatty acids. Since ligand selectivity was maintained between wild type and all mutants while ligand affinity was grossly diminished, we conclude that phenylalanine 57 is critical to the formation of the fatty acid/ALBP complex, but is uninvolved in determination of selectivity over the range of physiological ligands tested.
Simpson
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MA
Department of Biochemistry, University of Minnesota, St. Paul 55108, USA.
Bernlohr
D A
DA
D017667:Adipocytes / Q000378:metabolism*; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000235:genetics* / Q000378:metabolism*; D005456:Fluorescent Dyes; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D018996:Myelin P2 Protein / Q000235:genetics* / Q000378:metabolism; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D010649:Phenylalanine / Q000235:genetics*; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D002352:Carrier Proteins; C082667:Fabp5 protein, mouse; C086936:Fabp7 protein, mouse; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D005456:Fluorescent Dyes; D008024:Ligands; D018996:Myelin P2 Protein; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D010649:Phenylalanine; C027132:1-anilino-8-naphthalenesulfonate