Structural and functional characterization of the phosphorylated adipocyte lipid-binding protein (pp15)

Abstract

A substrate for the insulin receptor kinase in 3T3-L1 adipocytes has previously been identified as the adipocyte lipid-binding protein (ALBP, also known as aP2 or p15). We have characterized the effect of tyrosyl phosphorylation on ALBP structure and ligand-binding properties. Phosphorylated ALBP (phospho-ALBP) was isolated by a combination of gel filtration, anion exchange chromatography, and immunoaffinity chromatography on anti-phosphotyrosine agarose. Circular dichroic spectroscopy indicated that the phosphoprotein was similar in structure to native ALBP. Phospho-ALBP exhibited a slight decrease in calculated alpha-helical content which was compensated for by an increase in beta-sheet structure. The wavelength yielding maximum tryptophan fluorescence was unaltered by phosphorylation (334 +/- 1 nm). However, the concentration of guanidine HCl yielding 50% denaturation was 1.43 M for ALBP and 0.92 M for phospho-ALBP. The delta Goapp was 3.87 and 3.25 kcal mol-1 for ALBP and phospho-ALBP, respectively, suggesting that phosphorylation destabilized the protein. To assess the binding characteristics of the phosphoprotein, a long-chain fatty acid affinity column was synthesized to which native ALBP specifically bound. In contrast, phospho-ALBP showed little or no affinity for the column. Furthermore, phosphorylation virtually abolished binding of the fluorescent fatty acid analogue 12-(9-anthroyloxy)oleic acid. Fatty acid binding activity was recovered (approximately 60%) upon dephosphorylation with protein tyrosine phosphatase. The structural studies, coupled with the crystal structure of the apoprotein, indicate that the dramatic reduction in binding affinity is likely a result of steric hindrance in the binding cavity or of electrostatic interactions of the phosphoryl group with the fatty acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical and biophysical analysis of the intracellular lipid binding proteins of adipocytes

“…Relative stability of ALBP/aP2 and KLBP was assessed by monitoring the shift in maximum tryptophan emission wavelength as a function of increasing guanidine HCl concentration as previously described [14]. Denaturation curves were analyzed to determine Cm, the concentration of guanidine HCl at 50% denaturation, and free energies of unfolding were calculated by the linear extrapolation method of Pace [15].…”

X‐ray crystallographic analysis of adipocyte fatty acid binding protein (aP2) modified with 4‐hydroxy‐2‐nonenal

“…To optimize the purification seme, a long-chain fatty acid affinity column 29 was added in the purification procedure followed by 4-HNE modification in room temperature for 70 minutes. The sample was dialyzed and loaded onto the long-chain fatty acid affinity column to enrich for modified protein, which was eluted with 1 M NaCl.…”

“…The modified protein was applied to a long-chain acyl carboxylate affinity column and eluted with 1M NaCl. 29 Protein in the bound and unbound fractions was analyzed by mass spectrometry (Fig. 1) where covalent 4-HNE modification was accompanied by a $156 Da increase in the detected molecular mass of the protein.…”

Specificity determinants for lipids bound to β-barrel proteins

“…The midpoints of unfolding were determined from the denaturation curves. Gibb's free energy was determined by the ratio of denatured to native protein at each concentration of denaturant and then extrapolation to the zero-point concentration (37,38).…”