The helical domain of intestinal fatty acid binding protein is critical for collisional transfer of fatty acids to phospholipid membranes

Abstract

Fatty acid binding proteins (FABPs) exhibit a beta-barrel topology, comprising 10 antiparallel beta-sheets capped by two short alpha-helical segments. Previous studies suggested that fatty acid transfer from several FABPs occurs during interaction between the protein and the acceptor membrane, and that the helical domain of the FABPs plays an important role in this process. In this study, we employed a helix-less variant of intestinal FABP (IFABP-HL) and examined the rate and mechanism of transfer of fluorescent anthroyloxy fatty acids (AOFA) from this protein to model membranes in comparison to the wild type (wIFABP). In marked contrast to wIFABP, IFABP-HL does not show significant modification of the AOFA transfer rate as a function of either the concentration or the composition of the acceptor membranes. These results suggest that the transfer of fatty acids from IFABP-HL occurs by an aqueous diffusion-mediated process, i.e., in the absence of the helical domain, effective collisional transfer of fatty acids to membranes does not occur. Binding of wIFABP and IFABP-HL to membranes was directly analyzed by using a cytochrome c competition assay, and it was shown that IFABP-HL was 80% less efficient in preventing cytochrome c from binding to membranes than the native IFABP. Collectively, these results indicate that the alpha-helical region of IFABP is involved in membrane interactions and thus plays a critical role in the collisional mechanism of fatty acid transfer from IFABP to phospholipid membranes.

Figure 1

Effect of acceptor membrane concentration on AOFA transfer from FABP. Transfer of 1.5 μM 12AO from 15 μM wIFABP to EPC/NBD-PC SUV (•) and of 0.75 μM 12AO from 45 μM IFABP-HL to EPC/NBD-PC SUV (▿). Results are expressed relative to the 12AO transfer rates for 5:1 FABP/SUV, which were 0.38 ± 0.07 per sec for wIFABP and 1.25 ± 0.28 per sec for IFABP-HL. Averages of three different experiments ± SD are shown. A representative scan of experimental data is shown in the Inset (0.75 μM 12AO, 45 μM IFABP-HL, 225 μM EPC/NBD-PC SUV), with the rate of 1.35 per sec obtained by exponential fitting, as described in Materials and Methods.

Figure 2

Effect of vesicle charge on AOFA transfer from FABP. Transfer of 1.5 μM 12AO from 15 μM wIFABP to 150 μM EPC/NBD-PC SUV containing 25 mol % of PS or CL (solid bars) or 0.75 μM 12AO from 45 μM IFABP-HL (open bars) to 450 μM EPC/NBD-PC SUV containing 25 mol % of PS or CL. Results are expressed relative to the transfer rate of 12AO to EPC/NBD-PC membranes. Averages from three different experiments ± SD are shown.

Figure 3

Inhibition of cytochrome c binding to anionic membranes by FABP. (A) Increasing concentrations of cytochrome c were incubated with 12.5 μMSUV (EPC/EPE/CL/DPE, 64:10:25:1). The quenching of dansyl fluorescence is expressed relative to the fluorescence intensity of vesicles without cytochrome c. (B) 12.5 μM SUV containing 1% DPE were incubated with increasing concentrations of IFABP (•) or FABP-HL (▾) for 5 min, and 0.75 μM cytochrome c was then added as described in Materials and Methods. Results are expressed as the percent relative fluorescence intensity, where 100% represents the relative fluorescence intensity of SUV incubated in the presence of cytochrome c, but without FABP. Results are the average of three experiments ± SD.