FACTORS AFFECTING THE STABILITY AND CONFORMATION OF LOCUSTA-MIGRATORIA APOLIPOPHORIN-III

Apolipophorin III (apoLp-III) from the migratory locust, Locusta migratoria, represents the only full-length apolipoprotein whose three-dimensional structure has been solved. In the present study, spectroscopic methods have been employed to investigate the effects of deglycosylation (via endoglycosidase F treatment) and complexation with lipid on the stability and conformation of this protein. Addition of isolated lipid-free apoLp-III to sonicated vesicles of dimyristoylphosphatidylcholine (DMPC) resulted in the formation of relatively uniform disklike complexes with an average Stokes diameter of 13.5 nm. Flotation equilibrium experiments conducted in the analytical ultracentrifuge revealed a particle molecular mass of 588 500 Da. Chemical cross-linking and compositional analysis of apoLp-III.DMPC complexes indicated five apoLp-III molecules per disk and an overall DMPC:apoLp-III molar ratio of 122:1. Circular dichroism (CD) spectra of apoLp-III samples suggested a loss of alpha-helical structure upon deglycosylation, while complexation with DMPC did not significantly alter the helix content (estimated to be >75%). Fluorescence spectroscopy revealed that the apoLp-III tryptophan fluorescence emission maximum was blue-shifted from 347 to 332 and 321 nm upon deglycosylation and complexation with DMPC, respectively. In quenching experiments with native apoLp-III, tryptophan residues were shielded from the positively charged quencher, CsCl. Increased exposure to KI, CsCl, and acrylamide was observed upon deglycosylation, whereas complexation with DMPC yielded lower K(sv) values for KI and acrylamide and an increased value for CsCl versus native lipid-free apoLp-III. In guanidine hydrochloride denaturation studies monitored by CD or fluorescence, native, lipid-free apoLp-III displayed a denaturation midpoint of 0.60 M, and DELTAG(D)H2O = 5.37 kcal/mol was calculated. Deglycosylation decreased the stability of apoLp-III, while complexation with lipid stabilized its structure (increasing the denaturation midpoint substantially). The effect of solvent pH on the secondary structure stability of apoLp-III suggested that the protein is relatively resistant to pH-induced denaturation. This result is consistent with the concept that ion-pairing interactions between oppositely charged amino acid side chains may not play a significant role in stabilizing the lipid-free conformation of this protein. Near-UV CD spectra of apoLp-III revealed distinct peaks attributable to its two tryptophan and two phenylalanine residues. Deglycosylated apoLp-III possessed the same peaks, but these were reduced in magnitude. The spectra of apoLp-III.DMPC complexes also displayed extrema corresponding to tryptophan and phenylalanine residues, but these were reversed in sign and red-shifted. These data are consistent with a conformational change occurring upon the complexation of apoLp-III with lipid.