Shape and lipid-binding site of the nonspecific lipid-transfer protein (sterol carrier protein 2): a steady-state and time-resolved fluorescence study.
The nonspecific lipid-transfer protein (nsL-TP) from bovine liver was studied with time-resolved and steady-state fluorescence techniques. From the decay of the intrinsic tryptophanyl fluorescence, it was estimated that the rotational correlation time of nsL-TP is 15 ns. This parameter increased only slightly upon addition of an excess of negatively charged vesicles, indicating that the basic nsL-TP is not immobilized at the membrane surface under these conditions. Binding studies using fluorescent lipid analogues revealed that nsL-TP is able to extract sn-2-(pyrenehexanoyl) phosphatidylcholine and 1-palmitoyl-2-[3-(diphenylhexatrienyl) propionyl]-sn-3-phosphocholine (DPHp-PC) from a quenched donor vesicle. The fluorescence increase resulting from this binding was poorly quenched by either acrylamide or iodide. This indicates that nsL-TP shields the bound PC molecules from the aqueous environment. Time-resolved analysis of DPH fluorescence originating from DPHp-PC bound to nsL-TP yielded a rotational correlation time of 7.4 ns. This correlation time strongly suggests that the DPH moiety of the bound molecule is immobilized and that the nsL-TP/DPHp-PC complex is not attached to the donor vesicle. In view of the longer rotational correlation time obtained for the intrinsic tryptophanyl fluorescence, we conclude that nsL-TP is highly asymmetric. The data are consistent with a model in which the shape of nsL-TP is ellipsoidal with an axis ratio of 2.8. The implications for the mode of action of nsL-TP are discussed.
Gadella
T W
TW
Center for Biomembranes and Lipid Enzymology, State University of Utrecht, The Netherlands.
Bastiaens
P I
PI
Visser
A J
AJ
Wirtz
K W
KW
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry*; D002417:Cattle; D005454:Fluorescence Polarization; D008099:Liver / Q000737:chemistry; D008958:Models, Molecular; D010743:Phospholipids / Q000737:chemistry; D010940:Plant Proteins; D013261:Sterols / Q000378:metabolism; D014364:Tryptophan
D002352:Carrier Proteins; D010743:Phospholipids; D010940:Plant Proteins; D013261:Sterols; C072087:sterol carrier proteins; D014364:Tryptophan