Deletion of central alpha-helices in human apolipoprotein A-I: effect on phospholipid association.
In order to better understand the structure-function properties of apolipoprotein (apo) A-I, we have constructed and expressed three apoA-I mutants using a system previously described for the expression of human apolipoprotein A-I (Rec.-apoA-I). These mutants (corresponding to deletion of apoA-I residues 100-143, 122-165, 144-186) have been studied for their ability to form reconstituted apoA-I-containing lipoproteins (LpA-I) with POPC and DMPC, and for their structural and physical properties. Rec.- and native apoA-I can form homogeneous discoidal Lp2A-I over a wide range of POPC/apoA-I ratios [(20-130)/1] and exhibit sizes ranging from 9.5 to 10.5 nm. When recombined with varying POPC content [(20-130)/1, POPC/A-I)], the three mutants produce homogeneous discoidal Lp2A-I that contain a low POPC/A-I molar ratio [(20-40)/l for all mutants] and exhibit a nearly constant size [7.5-7.6 nm for delta (100-143) and 7.9-8.0 nm for the other two mutants]. Kinetics of association of these proteins with DMPC are similar for delta (100-143) and Rec.-apoA-I (t 1/2 of 4.0 and 4.4 min, respectively) but appear significantly reduced for delta (122-165) and delta (144-186) (t 1/2 of 7.5 and 6.9 min, respectively). While in the lipid-free form, all proteins have a similar thermodynamic stability with a very comparable free energy of unfolding (delta GD degree) for the alpha-helical structure, as determined by isothermal denaturation studies. delta-(100-143) has a significantly lower alpha-helical content (33%) as compared to the other proteins [40, 41, and 45% for Rec.-apoA-I. delta (122-165), and delta (144-186), respectively]. When associated to POPC, delta (122-165) and delta (144-186) have a higher alpha-helicity (63 and 63%) and an enhanced stability (2.5 and 2.3 kcal/mol, respectively) as compared to delta (100-143) (49% and 1.8 kcal/mol) and Rec.-apoA-I (52% and 1.9 kcal/mol). These results suggest that the amphipathic alpha-helices within residues 100-186 are directly involved in interactions with phospholipids. The helical region 100-121 appears to be more important to the stabilization of the lipid-apoprotein complex formed whereas helices within residues 122-186 appear to be critical to the initial rates of association of the apoprotein with DMPC. These data suggest that an important role of the central domain 100-186 may be to maintain the plasticity of apoA-I and its ability to form different classes of HDL particles. Therefore, it is likely that this region may also play an important role in the functional properties of this apoprotein.
Frank
P G
PG
Lipoproteins & Atherosclerosis Group, University of Ottawa Heart Institute, Ontario, Canada.
Bergeron
J
J
Emmanuel
F
F
Lavigne
J P
JP
Sparks
D L
DL
Denèfle
P
P
Rassart
E
E
Marcel
Y L
YL
D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002942:Circular Dichroism; D004134:Dimyristoylphosphatidylcholine; D004587:Electrophoresis, Agar Gel; D006801:Humans; D007700:Kinetics; D017270:Lipoprotein(a) / Q000031:analogs & derivatives / Q000737:chemistry / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis
D016632:Apolipoprotein A-I; D017270:Lipoprotein(a); D010743:Phospholipids; D011994:Recombinant Proteins; C078260:lipoprotein A-I; D004134:Dimyristoylphosphatidylcholine