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approach involves membrane protein purification and the assessment of lipid-
binding parameters in detergent micelles. These experimental methods may lead
to the revelation of a better mechanistic understanding of how certain lipid-binding
proteins are involved in LD formation and may also be used to better understand
other fundamental cellular processes, including lipid transport proteins or lipid flip-
pases. While certain species of TAG and DAG were used to assess lipid binding by
FIT2, a gamut of lipids with various degrees of unsaturation chain length and fatty
acid position on the glycerolipid backbone can be tested to determine the structural
specificity of protein-lipid interactions.
The yields that were obtained for the purified recombinant proteins discussed in
this chapter lend themselves to protein crystallography trials. Traditional crystallog-
raphy can be attempted using commercial kits in conjunction with a wide array of
conditions at a variety of temperatures. More recently, a novel crystallization
technique has been developed—lipidic mesophase crystallography—in which
membrane proteins in detergent micelles are mixed to homogeneity in monoolein
in order to generate an ordered cubic phase where the protein of interest is stabilized
and organized in a more natural membrane-like lipid bilayer reservoir when
compared to detergent micelles. This method has been used to determine the
structures of G-protein-coupled receptor-ligand complexes that were stabilized in
the active state with nanolid antibodies (
Caffrey, 2009; Caffrey & Cherezov,
2009; Rasmussen et al., 2011
).
We propose that the above-described purification techniques can be used to
purify perilipin family members and TAG-synthesizing proteins like DGAT family
members in an effort to reconstitute LD formation
in vitro
.
Acknowledgment
This work was supported, in whole or in part, by the Singapore Ministry of Health's National
Medical Research Council CBRG/0012/2012 (to D.L.S.), and the NIH MSTP Training Pro-
gram 5T32 GM 7288-37 (to D. A. G.).
References
