Monitoring glycolipid transfer protein activity and membrane interaction with the surface plasmon resonance technique.
The glycolipid transfer protein (GLTP) is a protein capable of binding and transferring glycolipids. GLTP is cytosolic and it can interact through its FFAT-like (two phenylalanines in an acidic tract) motif with proteins localized on the surface of the endoplasmic reticulum. Previous in vitro work with GLTP has focused mainly on the complete transfer reaction of the protein, that is, binding and subsequent removal of the glycolipid from the donor membrane, transfer through the aqueous environment, and the final release of the glycolipid to an acceptor membrane. Using bilayer vesicles and surface plasmon resonance spectroscopy, we have now, for the first time, analyzed the binding and lipid removal capacity of GLTP with a completely label-free technique. This technique is focused on the initial steps in GLTP-mediated transfer and the parameters affecting these steps can be more precisely determined. We used the new approach for detailed structure-function studies of GLTP by examining the glycolipid transfer capacity of specific GLTP tryptophan mutants. Tryptophan 96 is crucial for the transfer activity of the protein and tryptophan 142 is an important part of the proteins membrane interacting domain. Further, we varied the composition of the used lipid vesicles and gained information on the effect of membrane properties on GLTP activity. GLTP prefers to interact with more tightly packed membranes, although GLTP-mediated transfer is faster from more fluid membranes. This technique is very useful for the study of membrane-protein interactions and lipid-transfer rates and it can easily be adapted to other membrane-interacting proteins.
Ohvo-Rekilä
Henna
H
Biochemistry, Department of Biosciences, Åbo Akademi University, Turku, Finland. hohvo@abo.fi
Mattjus
Peter
P
D020816:Amino Acid Motifs; D001703:Biophysics; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006017:Glycolipids / Q000737:chemistry*; D008055:Lipids / Q000737:chemistry; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance / Q000379:methods*; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
D002352:Carrier Proteins; D006017:Glycolipids; D008055:Lipids; C030363:lipid transfer protein; D010649:Phenylalanine; D014364:Tryptophan