Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology.
A new approach for analyzing lipid-lipid transfer protein interactions is described. The transfer protein is genetically engineered for expression with a C-terminal biotinylated peptide extension (AviTag). This allows protein anchoring to a streptavidin-coated chip for surface plasmon resonance (SPR)-based assessment of lipid binding. Sterol carrier protein-2 (SCP-2), involved in the intracellular trafficking of cholesterol, fatty acids, and other lipids, was selected as the prototype. Biotinylated SCP-2 (bSCP-2) was expressed in Escherichia coli, purified to homogeneity by mutated streptavidin (SoftLink) affinity chromatography, and confirmed by mass spectrometry to contain one biotin group at the expected position. Intermembrane [(14)C]cholesterol transfer was strongly enhanced by bSCP-2, demonstrating that it was functional. Using bSCP-2 immobilized on a Biacore streptavidin chip, we determined on- and off-rate constants along with equilibrium dissociation constants for the following analytes: oleic acid, linoleic acid, cholesterol, and fluorophore (NBD)-derivatized cholesterol. The dissociation constant for NBD-cholesterol was similar to that determined by fluorescence titration for SCP-2 in solution, thereby validating the SPR approach. This method can be readily adapted to other transfer proteins and has several advantages over existing techniques for measuring lipid binding, including (i) the ability to study lipids in their natural states (i.e., without relatively large reporter groups) and (ii) the ability to measure on- and off- rate constants as well as equilibrium constants.
Kernstock
Robert M
RM
Department of Biochemistry, Medical College of Wisconsin, Milwaukee WI 53226, USA.
Girotti
Albert W
AW
D001692:Biological Transport; D015374:Biosensing Techniques / Q000379:methods*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism; D003001:Cloning, Molecular; D006801:Humans; D050356:Lipid Metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry*; D020349:Surface Plasmon Resonance / Q000379:methods*
D002352:Carrier Proteins; D011994:Recombinant Proteins; C030363:lipid transfer protein; C072087:sterol carrier proteins; D019809:Streptavidin; D002784:Cholesterol