A Plug-Based Microfluidic System for Dispensing Lipidic Cubic Phase (LCP) Material Validated by Crystallizing Membrane Proteins in Lipidic Mesophases.
This paper presents a plug-based microfluidic system to dispense nanoliter-volume plugs of Lipidic Cubic Phase (LCP) material and subsequently merge the LCP plugs with aqueous plugs. This system was validated by crystallizing membrane proteins in lipidic mesophases, including LCP. This system allows for accurate dispensing of LCP material in nanoliter volumes, prevents inadvertent phase transitions that may occur due to dehydration by enclosing LCP in plugs, and is compatible with the traditional method of forming LCP material using a membrane protein sample, as shown by the successful crystallization of bacteriorhodopsin from Halobacterium salinarum. Conditions for the formation of LCP plugs were characterized and presented in a phase diagram. This system was also implemented using two different methods of introducing the membrane protein: 1) the traditional method of generating the LCP material using a membrane protein sample and 2) Post LCP-formation Incorporation (PLI), which involves making LCP material without protein, adding the membrane protein sample externally to the LCP material, and allowing the protein to diffuse into the LCP material or into other lipidic mesophases that may result from phase transitions. Crystals of bacterial photosynthetic reaction centers from Rhodobacter sphaeroides and Blastochloris viridis were obtained using PLI. The plug-based, LCP-assisted microfluidic system, combined with the PLI method for introducing membrane protein into LCP, should be useful for minimizing consumption of samples and broadening the screening of parameter space in membrane protein crystallization.
Li
Liang
L
Department of Chemistry and Institute for Biophysical Dynamics The University of Chicago, 929 East 57 Street, Chicago, IL, 60637.
Fu
Qiang
Q
Kors
Christopher A
CA
Stewart
Lance
L
Nollert
Peter
P
Laible
Philip D
PD
Ismagilov
Rustem F
RF
J Bacteriol. 1988 Jan;170(1):320-9
3257209
Biophys J. 2001 Jul;81(1):225-42
11423409
Structure. 2008 Jul;16(7):1003-9
18611373
Anal Chem. 2006 Jul 15;78(14):4839-49
16841902
J Mol Biol. 2003 Aug 15;331(3):681-92
12899837
Biochemistry. 1996 Aug 20;35(33):10763-75
8718867
J Mol Biol. 2006 Apr 14;357(5):1605-18
16490208
J Am Chem Soc. 2007 Jul 18;129(28):8825-35
17580868
Acta Crystallogr D Biol Crystallogr. 2004 Oct;60(Pt 10):1795-807
15388926
Anal Chem. 1998 Dec 1;70(23):4974-84
21644679
Curr Opin Struct Biol. 2005 Oct;15(5):548-55
16154351
FEBS Lett. 1985 May 20;184(2):231-6
3888667
Biophys J. 2000 Jul;79(1):394-405
10866965
Curr Opin Struct Biol. 1997 Oct;7(5):697-701
9345629
J Bacteriol. 1987 Mar;169(3):1341-4
3818549
Anal Chem. 2005 Feb 1;77(3):785-96
15679345
Biochemistry. 2002 May 14;41(19):5998-6007
11993994
Curr Opin Struct Biol. 2000 Aug;10(4):486-97
10981640
Lab Chip. 2004 Aug;4(4):316-21
15269797
J Am Chem Soc. 2007 Jan 24;129(3):454-5
17226984
Electrophoresis. 2000 Jan;21(1):27-40
10634468
Lab Chip. 2006 Dec;6(12):1484-6
17203151
Angew Chem Int Ed Engl. 2006 Nov 13;45(44):7336-56
17086584
J Am Chem Soc. 2003 Sep 17;125(37):11170-1
16220918
Science. 2008 Nov 21;322(5905):1211-7
18832607
Science. 2007 Nov 23;318(5854):1258-65
17962520
Cryst Growth Des. 2009 Jun 3;9(6):2566-2569
20161169
Angew Chem Int Ed Engl. 2003 Feb 17;42(7):768-72
12596195
J Mol Biol. 2006 Nov 17;364(1):44-53
17005199
Methods. 2004 Nov;34(3):348-53
15325652
Proc Natl Acad Sci U S A. 2006 Dec 19;103(51):19243-8
17159147
Biochemistry. 1992 Oct 27;31(42):10345-55
1420154