Faculty Collaboration Database

Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology. Anal Biochem 2007 Jun 01;365(1):111-21

Date

03/23/2007

Pubmed ID

17376396

Pubmed Central ID

PMC1975857

DOI

10.1016/j.ab.2007.02.018

Scopus ID

2-s2.0-34247228720 (requires institutional sign-in at Scopus site)   11 Citations

Abstract

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.

Author List

Kernstock RM, Girotti AW

Author

Albert Girotti PhD, MS Emeritus Professor in the Biochemistry department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Biological Transport
Biosensing Techniques
Carrier Proteins
Cholesterol
Cloning, Molecular
Humans
Lipid Metabolism
Protein Binding
Recombinant Proteins
Streptavidin
Surface Plasmon Resonance