β-Conglycinin, one of the dominant storage proteins of soybean, is a trimer composed of three subunits, A, A′ and β. All subunits are N-glycosylated and A and A′ contain extension regions in addition to the core regions common to all subunits. Non-glycosylated individual subunits and deletion mutants (A c and A′ c ) lacking the extension regions of A and A′ were expressed in Escherichia coli. All recombinant proteins were purified to near homogeneity and appeared to have the correct conformation, as judged by CD, density-gradient centrifugation and gel-filtration profiles, indicating that the N-linked glycans and extension regions are not essential for the folding and the assembly into trimers of β-conglycinin. Densitygradient centrifugation, gel-filtration and differential scanning calorimetry profiles of the recombinant proteins and the native β-conglycinin indicated that the N-linked glycans and extension regions contribute to the dimension of β-conglycinin but not to the density and the thermal stability. Comparing the solubilities of the individual subunits with those of deletion mutants, only the A and A′ subunits were soluble at lower ionic strength (µ Ͻ 0.25) at around the pH value of the endoplasmic reticulum. This suggests that the extension regions play an important role in the prevention of aggregation in the endoplasmic reticulum in analogy with the N-linked glycans.

No abstract

Nanodiscs are phospholipid-protein complexes which are relevant to nascent high-density lipoprotein and are applicable as a drug carrier and a tool to immobilize membrane proteins. We evaluated the structure and dynamics of the nanoparticles consisting of dimyristoylphosphatidylcholine (DMPC) and apolipoprotein A-I (apoA-I) with small-angle neutron scattering (SANS) and fluorescence methods and compared them with static/dynamic properties for large unilamellar vesicles. SANS revealed that the nanodisc includes a lipid bilayer with a thickness of 44 A and a radius of 37 A, in which each lipid occupies a smaller area than the reported molecular area of DMPC in vesicles. Fluorescence measurements suggested that DMPC possesses a lower entropy in nanodiscs than in vesicles, because apoA-I molecules, which surround the bilayer, force closer lipid packing, but allow water penetration to the acyl chain ends. Time-resolved SANS experiments revealed that nanodiscs represent a 20-fold higher lipid transfer via an entropically favorable process. The results put forward a conjunction of static/dynamic properties of nanodiscs, where the entropic constraints are responsible for the accelerated desorption of lipids.