(650.9) Fatty Acid-Binding Proteins Respond Differently When Interacting with Endogenous Cannabinoids under High Pressure

Poster Board Number: A119

Irving Rosario Jr (CUNY City College of New York, CUNY City College of New York, CUNY City College of New York), Roksana Azad (CUNY Advanced Science Research Center, CUNY Advanced Science Research Center), May Poh Lai (CUNY Institute for Macromolecular Assemblies, CUNY Institute for Macromolecular Assemblies, CUNY Institute for Macromolecular Assemblies), Donald Gagne (CUNY Advanced Science Research Center), Hsin Wang (CUNY City College of New York, CUNY City College of New York), Francine Katz (CUNY City College of New York, CUNY City College of New York), Kevin Gardner (CUNY Advanced Science Research Center, CUNY Advanced Science Research Center, CUNY Advanced Science Research Center), Ruth Stark (CUNY City College of New York, CUNY City College of New York, CUNY City College of New York)

Liver-type and intestinal fatty acid-binding proteins (LFABP and IFABP, respectively) are both located in enterocytes, but their different functions are demonstrated by phenotypic differences observed in knockout mice for either protein. Though the two proteins are structurally similar, they differ significantly in their ligand-binding cavities. The region of a protein with the greatest conformational variability is often found in the area surrounding the ligand-binding cavity to enable an induced fit to a specific substrate. We used solution NMR spectroscopy while applying increasing pressure to investigate the interaction of these two proteins with ligand, in the hopes of capturing transient intermediate conformations of the protein and to identify potential on-pathway states for substrate binding that would not otherwise be seen under standard conditions.

Our initial experiments showed apo- and ligand-bound IFABP remains stable under increasing pressure, up to 2500 bar. In contrast, apo-LFABP and LFABP with anandamide completely unfolds at 1750 bar. Interestingly, the presence of oleate and arachidonic acid stabilizes LFABP, making it resistant to unfolding at high pressure. Our results show that this stabilization is ligand specific, correlating with the ligand binding affinities we determined previously. This trend suggests that specific protein/ligand interactions within the cavity are required for stability.

To make a more detailed analysis of which residues are involved in binding, we generated plots of the pressure-dependence of backbone 15N and 1H chemical shifts of LFABP and IFABP in either apo- or holo- form. Two regions of the protein were most responsive to changes in pressure as assessed by the nonlinear chemical shifts changes: i) the conformationally flexible portal and ii) a region of the ligand-binding cavity we had previously reported as involved in the specificity of binding endocannabinoid ligands. This finding is consistent with the requirement of the conformational variability in these regions for ligand binding. We are currently performing a comprehensive analysis of these effects for the interaction of both proteins with a range of structurally related endogenous cannabinoids.

NIH T32 GM136499
NSF MCB-1818148
NIH R01 GM106239