Interfacial exclusion pressure determines the ability of apolipoprotein A-IV truncation mutants to activate cholesterol ester transfer protein.
We used a panel of recombinant human apolipoprotein (apo) A-IV truncation mutants, in which pairs of 22-mer alpha-helices were sequentially deleted along the primary sequence, to examine the impact of protein structure and interfacial activity on the ability of apoA-IV to activate cholesterol ester transfer protein. Circular dichroism and fluorescence spectroscopy revealed that the secondary structure, conformation, and molecular stability of recombinant human apoA-IV were identical to the native protein. However, deletion of any of the alpha-helical domains in apoA-IV disrupted its tertiary structure and impaired its molecular stability. Surprisingly, determination of the water/phospholipid interfacial exclusion pressure of the apoA-IV truncation mutants revealed that, for most, deletion of amphipathic alpha-helical domains increased their affinity for phospholipid monolayers. All of the truncation mutants activated the transfer of fluorescent-labeled cholesterol esters between high and low density lipoproteins at a rate higher than native apoA-IV. There was a strong positive correlation (r = 0.790, p = 0.002) between the rate constant for cholesterol ester transfer and interfacial exclusion pressure. We conclude that molecular interfacial exclusion pressure, rather than specific helical domains, determines the degree to which apoA-IV, and likely other apolipoproteins, facilitate cholesterol ester transfer protein-mediated lipid exchange.
Weinberg
Richard B
RB
Department of Internal Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA. weinberg@wfubmc.edu
Anderson
Rachel A
RA
Cook
Victoria R
VR
Emmanuel
Florence
F
Denèfle
Patrice
P
Tall
Alan R
AR
Steinmetz
Armin
A
D001054:Apolipoproteins A / Q000235:genetics*; D002788:Cholesterol Esters / Q000378:metabolism*; D002942:Circular Dichroism; D017353:Gene Deletion; D006801:Humans; D050356:Lipid Metabolism; D008074:Lipoproteins / Q000378:metabolism; D009154:Mutation; D011312:Pressure; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D001054:Apolipoproteins A; D002788:Cholesterol Esters; D008074:Lipoproteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C022205:apolipoprotein A-IV