Differential Lipid Binding of Truncation Mutants of Galleria mellonella Apolipophorin III

Abstract

Apolipophorin III (apoLp-III) is a prototype exchangeable apolipoprotein that is amenable to structure-function studies. The protein folds as a bundle of five amphipathic alpha-helices and undergoes a dramatic conformational change upon lipid binding. Recently, we have shown that a truncation mutant of Galleria mellonella apoLp-III comprising helices 1-3 is stable in solution and able to bind to lipid surfaces [Dettloff, M., Weers, P. M. M., Niere, M., Kay, C. M., Ryan, R. O., and Wiesner, A. (2001) Biochemistry 40, 3150-3157]. To investigate the role of the C-terminal helices in apoLp-III structure and function, two additional 3-helix mutants were designed: a core fragment comprising helix (H) 2-4, and a C-terminal fragment (H3-5). Each truncation mutant retained the ability to associate spontaneously with dimyristoylphosphatidylcholine (DMPC) vesicles, transforming them into discoidal complexes. The rate of apolipoprotein-dependent DMPC vesicle transformation decreased in the order H1-3 > H2-4 > H3-5. Truncation of two helices led to a significant decrease in alpha-helical content in buffer in each case, from 86% (wild-type) to 50% (H1-3), 28% (H2-4), and 24% alpha-helical content (H3-5). On the other hand, trifluoroethanol or complexation with DMPC induced the truncation mutants to adopt a high alpha-helical structure similar to that of wild-type protein (84-100% alpha-helical structure). ApoLp-III(H1-3) and apoLp-III(H2-4), but not apoLp-III(H3-5), were able to prevent phospholipase-C-induced low density lipoprotein aggregation, indicating that interaction of the C-terminal fragment with spherical lipoprotein surfaces was impaired. As lipoprotein binding is significantly affected and DMPC transformation rates are relatively slow upon removal of N-terminal helices, the data indicate that structural elements necessary for lipid interaction reside in the N-terminal part of the protein.

Modification by Acrolein, a Component of Tobacco Smoke and Age-Related Oxidative Stress, Mediates Functional Impairment of Human Apolipoprotein E

“…This ability has been used as a reliable and sensitive assay to evaluate lipid binding ability of apolipoproteins (30,31) in terms of the initial kinetics of transformation. Briefly, 1 ml TBS was added to a thin film of 5 mg DMPC, followed by vigorous vortexing to generate multi lamellar vesicles (MLVs).…”

Apolipophorin III: Lipopolysaccharide binding requires helix bundle opening

“…Escherichia coli BL21 (DE3) cells were transformed with the mutated plasmids, and cells were grown in Minimal Medium as described [23]. Since apoLp-III escaped the bacteria and appeared in the culture medium, cells were removed by centrifugation and the supernatant was collected and concentrated to 20 mL by tangential flow filtration with a 10K Minimate cassette membrane (Pall Life Sciences, Ann Arbor, MI).…”

“…Since apoLp-III escaped the bacteria and appeared in the culture medium, cells were removed by centrifugation and the supernatant was collected and concentrated to 20 mL by tangential flow filtration with a 10K Minimate cassette membrane (Pall Life Sciences, Ann Arbor, MI). ApoLp-III was further purified by G-75 gelfiltration chromatography and reversed-phase HPLC (Beckman system gold, C8 Zorbax semi preparative column) using a linear gradient of acetonitrile in water containing 0.05% trifluoroacetic acid [23]. The protein was freeze-dried and stored at -20 °C.…”

“…Unilamellar vesicles of DMPC (Avanti Polar Lipids, Birmingham, AL) were prepared in PBS by extrusion through 200 nm filters as described previously [23]. Phospholipid (250 μg) and protein (250 μg) were equilibrated at 24 °C, mixed (500 μl final volume), and the change in absorbance (325 nm) was measured with a Shimadzu spectrophotometer maintaining the incubation temperature with a Peltier controlled cell holder at 24 °C.…”

“…ApoLp-III has the ability to transform DMPC vesicles into much smaller discoidal complexes, a characteristic shared with other exchangeable apolipoproteins [8]. The transformation of unilamellar vesicles (200 nm in size) into discs (18 nm diameter, estimated by electron microscopy) is a spontaneous process at the lipid transition temperature, which is 24 °C for DMPC [23]. The transformation can be monitored spectrophotometrically as the turbid phospholipid suspension becomes transparent during formation of the much smaller discs.…”