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


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. Study holds ProTherm entries: 5207, 5208, 5209 Extra Details: lipid-free state; alpha-helical content; secondary structure;,hydrophobic stacking interaction; charge-charge interactions

Submission Details

ID: oZZ5GEu43

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:29 p.m.

Version: 1

Publication Details
Ryan RO;Oikawa K;Kay CM,J. Biol. Chem. (1993) Conformational, thermodynamic, and stability properties of Manduca sexta apolipophorin III. PMID:8420928
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant PDB Entries

Structure ID Release Date Resolution Structure Title

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Apolipophorin-3 P13276 APL3_MANSE