Modification of the adipocyte lipid binding protein by sulfhydryl reagents and analysis of the fatty acid binding domain.
The adipocyte lipid binding protein (ALBP) is a member of a multigene family of low molecular weight proteins which stoichiometrically and saturably bind hydrophobic ligands and presumably facilitate intracellular lipid metabolism. To probe the structure-function relationship of the binding domain of ALBP, chemical modification has been employed. Modification of the two cysteinyl residues of ALBP (Cys1 and Cys117) with a variety of sulfhydryl reagents decreased the apparent affinity for oleic acid in the following order of effectiveness: methyl methanethiosulfonate much much less than p-(chloromercuri)benzenesulfonic acid less than N-ethylmaleimide (NEM) = 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Thiol titration of ALBP with DTNB in the presence of bound oleate resulted in the modification of a single cysteinyl residue. The oleate-protected cysteine was identified as Cys117 by modification with a combination of reversible (DTNB) and irreversible (NEM) sulfhydryl reagents in the presence or absence of saturating oleic acid. Cys117-NEM ALBP exhibited a large decrease in binding affinity while Cys1-NEM ALBP exhibited normal binding properties. Neither the modification of ALBP with NEM nor the addition of oleic acid had a significant effect on protein structure, as judged by circular dichroic analysis. These results suggest that Cys117 of ALBP resides in the ligand binding domain and that site-specific modification can be utilized to assess the conformational flexibility of the binding cavity.
Buelt
M K
MK
Department of Biochemistry, University of Minnesota, St. Paul 55108.
Bernlohr
D A
DA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D004228:Dithionitrobenzoic Acid; D005033:Ethylmaleimide; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds
D002352:Carrier Proteins; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D009363:Neoplasm Proteins; D009829:Oleic Acids; D013438:Sulfhydryl Compounds; D019301:Oleic Acid; D004228:Dithionitrobenzoic Acid; D005033:Ethylmaleimide