Conformational, thermodynamic, and stability properties of Manduca sexta apolipophorin III

Abstract

Apolipophorin III (apoLp-III) is a major protein in hemolymph of adult Manduca sexta. Although it normally exists in a lipid-free state, during sustained flight, apoLp-III functions as an apolipoprotein, reversibly associating with the surface of lipoprotein particles. In an effort to gain a fuller understanding of this dual existence, we have investigated its solution properties using spectroscopic methods. The UV absorption spectrum of apoLp-III is distinctive owing to the absence of tryptophan and the presence of a single tyrosine residue. Circular dichroism experiments revealed an enhancement of apoLp-III alpha-helical content when spectra were obtained in 50% trifluoroethanol versus aqueous buffer. The helical content in buffer was unaffected by protein concentration, suggesting that apoLp-III exists in solution as a monomeric species. At pH values > 10 and < 4, there was a marked loss of helical content. Increasing the temperature of apoLp-III solutions also caused a loss of secondary structure, with a temperature-induced denaturation midpoint of 52 degrees C. Upon recooling of heat-denatured apoLp-III, approximately 95% of the secondary structure was restored. In guanidine HCl denaturation studies monitored by CD, a 50% transition midpoint of 0.355 M was determined, corresponding to a delta GDH2O of 1.29 kcal/mol. Fluorescence studies indicated that guanidine HCl induced an enhancement of tyrosine fluorescence emission at 300 nm when excited at 277 nm. In native apoLp-III, we propose that tyrosine fluorescence is quenched to a large extent due to a hydrophobic stacking interaction of its side chain with that of a neighboring phenylalanine residue. delta GDH2O was determined from the fluorescence data to be 2.1 kcal/mol, with a transition midpoint occurring at 0.25 M guanidine HCl. The lower concentration of guanidine HCl required to induce half-maximal tyrosine fluorescence enhancement versus the transition midpoint detected by CD may be a reflection of the fact that this residue is located near the COOH-terminal end of the protein and as such may be more susceptible to denaturation. The results presented indicate that apoLp-III assumes a relatively labile conformation in solution that appears to be partially stabilized by side chain charge-charge interactions within predicted alpha-helical segments.