Lipid cubic phase as a membrane mimetic for integral membrane protein enzymes
File Type:
PDFItem Type:
Journal ArticleDate:
2011Access:
OpenAccessCitation:
Dianfan Li, Martin Caffrey, Lipid cubic phase as a membrane mimetic for integral membrane protein enzymes, PNAS, 108, 21, 2011, 8639-8644Download Item:
Abstract:
The lipidic cubic mesophase has been used to crystallize important membrane proteins for high-resolution structure determination. To date, however, no integral membrane enzymes have yielded to this method, the in meso. For a crystal structure to be meaningful the target protein must be functional. Using the in meso method with a membrane enzyme requires that the protein is active in the mesophase that grows crystals. Because the cubic phase is sticky and viscous and is bicontinuous topologically, quantitatively assessing enzyme activity in meso is a challenge. Here, we describe a procedure for characterizing the catalytic properties of the integral membrane enzyme, diacylglycerol kinase, reconstituted into the bilayer of the lipidic cubic phase. The kinase activity of this elusive crystallographic target was monitored spectrophotometrically using a coupled assay in a high-throughput, 96-well plate format. In meso, the enzyme exhibits classic Michaelis?UOMenten kinetics and works with a range of lipid substrates. The fact that the enzyme and its lipid substrate and product remain confined to the porous mesophase while its water-soluble substrate and product are free to partition into the aqueous bathing solution suggests a general and convenient approach for characterizing membrane enzymes that function with lipids in a membrane-like environment. The distinctive rheology of the cubic phase means that a procedural step to physically separate substrate from product is not needed. Because of its open, bicontinuous nature, the cubic phase offers the added benefit that the protein is accessible for assay from both sides of the membrane.
Sponsor
Grant Number
National Institutes of Health (NIH)
U54GM094599
National Institutes of Health (NIH)
P50GM073210
National Institutes of Health (NIH)
GM75915
European Commission
CM0902
Science Foundation Ireland (SFI)
07/IN.1/B1836
Author's Homepage:
http://people.tcd.ie/mcaffreDescription:
PUBLISHED
Author: CAFFREY, MARTIN; LI, DIANFAN
Type of material:
Journal ArticleCollections
Series/Report no:
PNAS108
21
Availability:
Full text availableKeywords:
Biochemistry, coupled enzyme assayDOI:
http://dx.doi.org/10.1073/pnas.1101815108Metadata
Show full item recordLicences: