Characterization of the lipid and protein organization in HBsAg viral particles by steady-state and time-resolved fluorescence spectroscopy.
Hepatitis B surface antigen (HBsAg) particles, produced in the yeast Hansenula polymorpha, are 20 nm particles, composed of S surface viral proteins and host-derived lipids. Since the detailed structure of these particles is still missing, we further characterized them by fluorescence techniques. Fluorescence correlation spectroscopy indicated that the particles are mainly monomeric, with about 70 S proteins per particle. The S proteins were characterized through the intrinsic fluorescence of their thirteen Trp residues. Fluorescence quenching and time-resolved fluorescence experiments suggest the presence of both low emissive embedded Trp residues and more emissive Trp residues at the surface of the HBsAg particles. The low emission of the embedded Trp residues is consistent with their close proximity in alpha-helices. Furthermore, S proteins exhibit restricted movement, as expected from their tight association with lipids. The lipid organization of the particles was studied using viscosity-sensitive DPH-based probes and environment sensitive 3-hydroxyflavone probes, and compared to lipid vesicles and low density lipoproteins (LDLs), taken as models. Like LDLs, the HBsAg particles were found to be composed of an ordered rigid lipid interface, probably organized as a phospholipid monolayer, and a more hydrophobic and fluid inner core, likely composed of triglycerides and free fatty acids. However, the lipid core of HBsAg particles was substantially more polar than the LDL one, probably due to its larger content in proteins and its lower content in sterols. Based on our data, we propose a structural model for HBsAg particles where the S proteins deeply penetrate into the lipid core.
Greiner
Vanille J
VJ
Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de pharmacie, Illkirch, France.
Egelé
Caroline
C
Oncul
Sule
S
Ronzon
Frédéric
F
Manin
Catherine
C
Klymchenko
Andrey
A
Mély
Yves
Y
D005456:Fluorescent Dyes; D006514:Hepatitis B Surface Antigens / Q000737:chemistry*; D008055:Lipids / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014764:Viral Proteins / Q000737:chemistry*
D005456:Fluorescent Dyes; D006514:Hepatitis B Surface Antigens; D008055:Lipids; D014764:Viral Proteins