Equilibrium and dynamic spectroscopic studies of the interaction of monomeric β-lactoglobulin with lipid vesicles at low pH.
β-Lactoglobulin (βLG) is a member of the lipocalin protein family that changes structure upon interacting with anionic surfactants and lipid vesicles under higher-pH conditions at which βLG is dimeric. In this study, a β-sheet to α-helix transformation was also observed for monomeric βLG obtained at pH 2.6 when it was mixed with small unilamellar vesicles (SUVs) of zwitterionic lipids, but being mixed with anionic lipids produced little change. The dynamics and extent of this change were quite dependent on the lipid character, phase, and vesicle size. With 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), at ~50 °C and pH 2.6, the βLG converted to a substantially helical form upon addition of ~10 mM lipid in a two-step kinetic process having time constants of ~1 and ~25 h, as monitored by circular dichroism (CD). Fluorescence changes were simpler but implied a rapid initial change in the Trp environments followed by a slower process paralleling the change in secondary structure. Polarization attenuated total reflectance Fourier transform infrared results indicate the formed helices are at least partially inserted into the lipid bilayer and the sheet segments are on the surface. Thermal behavior showed that the secondary structure of the lipid-bound βLG had two phases, the first being characteristic of the protein-lipid vesicle interaction and the second following the DSPC phase change after which the protein apparently dissociated from the vesicle. Large unilamellar vesicles had a weaker interaction, as judged by CD, which may correlate to the partial exposure of the hydrophobic parts of the SUV bilayer. Other zwitterionic lipids bound βLG with much slower kinetics and often required sonication to induce interaction, but these also showed dissociation upon lipid phase change. These thermal and kinetic behaviors suggest a mechanism for the interaction of monomeric βLG with zwitterionic lipids different from that seen previously for the dimeric form.
Department of Chemistry, University of Illinois at Chicago , 845 West Taylor Street (m/c 111), Chicago, Illinois 60607-7061, United States.
Ge
G
Zhang
Timothy A
TA
Keiderling
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000737:chemistry*; D010713:Phosphatidylcholines / Q000737:chemistry*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary
D007782:Lactoglobulins; D010713:Phosphatidylcholines