Cholesterol interaction with recombinant human sterol carrier protein-2.
The interaction of human recombinant sterol carrier protein-2 (SCP-2) with sterols was examined. Two independent ligand binding methods, Lipidex 1000 binding of [3H]cholesterol and a fluorescent dehydroergosterol binding assay, were used to determine the affinity of SCP-2 for sterols. Binding analysis indicated SCP-2 bound [3H]cholesterol and dehydroergosterol with a Kd of 0.3 and 1.7 microM, respectively, and suggested the presence of a single binding site. Phase fluorometry and circular dichroism were used to characterize the SCP-2 sterol binding site. Alterations in dehydroergosterol lifetime, SCP-2 tryptophan lifetime, and SCP-2 tryptophan quenching by acrylamide upon cholesterol binding demonstrated a shielding of the SCP-2 tryptophan from the aqueous solvent by bound sterol. Differential polarized phase fluorometry revealed decreased SCP-2 tryptophan rotational correlation time upon cholesterol binding. Circular dichroism of SCP-2 indicated that cholesterol elicited a small decrease in SCP-2 alpha helical content. The data suggest that SCP-2 binds sterols with affinity consistent with a lipid transfer protein that may act either as an aqueous carrier or at a membrane surface to enhance sterol desorption.
Colles
S M
SM
Department of Pharmacology and Cell Biophysics, University of Cincinnati Medical Center, Ohio 45267-0004, USA.
Woodford
J K
JK
Moncecchi
D
D
Myers-Payne
S C
SC
McLean
L R
LR
Billheimer
J T
JT
Schroeder
F
F
D020106:Acrylamide; D000178:Acrylamides / Q000737:chemistry; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D002942:Circular Dichroism; D004347:Drug Interactions; D004875:Ergosterol / Q000031:analogs & derivatives / Q000378:metabolism; D005470:Fluorometry; D006801:Humans; D008861:Microsomes / Q000378:metabolism; D010940:Plant Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D013261:Sterols / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism
D000178:Acrylamides; D002352:Carrier Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D013261:Sterols; C072087:sterol carrier proteins; C010905:dehydroergosterol; D020106:Acrylamide; D014364:Tryptophan; D002784:Cholesterol; D004875:Ergosterol