FATTY-ACID INTERACTIONS WITH A HELIX-LESS VARIANT OF INTESTINAL FATTY-ACID-BINDING PROTEIN

Intestinal fatty acid-binding protein (I-FABP) binds a single molecule of long-chain fatty acid in an enclosed cavity surrounded by two anti parallel beta-sheets. The structure also contains two short alpha-heli ces which form a cap over one end of the binding cavity adjacent to th e methyl terminus of the fatty acid. In this study, we employed a heli x-less variant of I-FABP known as Delta 17-SG [Kim, K., Cistola, D. P. , & Frieden, C. (1996) Biochemistry 35, 7553-7558] to investigate the role of the helical region in maintaining the integrity of the binding cavity and mediating the acquisition of ligand. Fluorescence and NMR experiments were used to characterize the energetic, structural, and k inetic properties of fatty acid binding to this variant, and the resul ts were compared and contrasted with those of wild-type I-FABP and a s ingle-site mutant, R106T. Remarkably, oleate bound to Delta 17-SG with a dissociation constant of 4.5 mu M, a value comparable to that for R 106T and approximately 20-100-fold higher than that for wild-type I-FA BP. Heteronuclear two-dimensional NMR spectra for [2-C-13]palmitate co mplexed with Delta 17-SG revealed a pattern nearly identical to that o bserved for the wild-type protein, but distinct from that for R106T. I n addition, the ionization behavior of bound [1-C-13]palmitate and the nearest neighbor patterns for [2-C-13]palmitate derived from C-13-fil tered NOESY experiments were very similar for Delta 17-SG and the wild -type protein. These results implied that the fatty acid-protein inter actions characteristic of the carboxyl end of the fatty acid binding c avity in the wild-type protein were essentially intact in the helix-le ss variant. In contrast, C-13-filtered NOESY spectra of [16-C-13]palmi tate bound to Delta 17-SG indicated that the fatty acid-protein intera ctions at the methyl end of the binding cavity were disrupted. As dete rmined by stopped-flow fluorescence, the observed ligand association r ates for both Delta 17-SG and wild-type I-FABP increased with increasi ng oleate concentration, but only the wild-type protein exhibited a li miting value of 1000 s(-1). This rate-limiting process was interpreted as a conformational change involving the helical region that allows t he ligand access to the internal cavity. Simulation and fitting of the kinetic results yielded ligand association rates for Delta 17-SG and wild-type I-FABP that were comparable. However, the dissociation rate for wild-type protein was 16-fold lower than that for Delta 17-SG. We conclude that the alpha-helices of I-FABP are not required to maintain the integrity of the fatty acid binding cavity but may serve to regul ate the affinity of fatty acid binding by selectively altering the dis sociation rate constant. In this manner, conformational changes involv ing the alpha-helical domain may help control the transfer of fatty ac ids within the cell.