The University of Dublin | Trinity College -- Ollscoil Átha Cliath | Coláiste na Tríonóide
Trinity's Access to Research Archive
Home :: Log In :: Submit :: Alerts ::

TARA >
School of Biochemistry & Immunology >
Biochemistry >
Biochemistry (Scholarly Publications) >

Please use this identifier to cite or link to this item: http://hdl.handle.net/2262/61017

Title: Lipid cubic phase as a membrane mimetic for integral membrane protein enzymes
Author: LI, DIANFAN
CAFFREY, MARTIN
Sponsor: 
Name Grant Number
U54GM094599
P50GM073210
GM75915
CM0902
07/IN.1/B1836

Author's Homepage: http://people.tcd.ie/mcaffre
http://people.tcd.ie/lidi
Keywords: Biochemistry
coupled enzyme assay
Issue Date: 2011
Publisher: National Academy of Sciences
Citation: Dianfan Li, Martin Caffrey, Lipid cubic phase as a membrane mimetic for integral membrane protein enzymes, PNAS, 108, 21, 2011, 8639-8644
Series/Report no.: PNAS;
108;
21;
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‰ÛÒMenten 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.
Description: PUBLISHED
URI: http://hdl.handle.net/2262/61017
Related links: http://dx.doi.org/10.1073/pnas.1101815108
Access: OpenAccess
Appears in Collections:Biochemistry (Scholarly Publications)

Files in This Item:

File Description SizeFormat
Lipid cubic phase as a membrane mimetic for integral membrane protein enzymes.pdfPublished (publisher's copy) - Peer Reviewed863.75 kBAdobe PDFView/Open


This item is protected by original copyright


Please note: There is a known bug in some browsers that causes an error when a user tries to view large pdf file within the browser window. If you receive the message "The file is damaged and could not be repaired", please try one of the solutions linked below based on the browser you are using.

Items in TARA are protected by copyright, with all rights reserved, unless otherwise indicated.

 

Valid XHTML 1.0! DSpace Software Copyright © 2002-2010  Duraspace - Feedback