Skip to main content

A plug-based microfluidic system for dispensing lipidic cubic phase (LCP) material validated by crystallizing membrane proteins in lipidic mesophases

Microfluidics and Nanofluidics volume 8, pages 789–798 (2010)Cite this article

Abstract

This article 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.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  • Ai X, Caffrey M (2000) Membrane protein crystallization in lipidic mesophases: detergent effects. Biophys J 79(1):394–405

    Article  Google Scholar 

  • Caffrey M (2000) A lipid’s eye view of membrane protein crystallization in mesophases. Curr Opin Struct Biol 10(4):486–497

    Article  Google Scholar 

  • Cherezov V, Caffrey M (2006) Picolitre-scale crystallization of membrane proteins. J Appl Crystallogr 39:604–606

    Article  Google Scholar 

  • Cherezov V, Fersi H, Caffrey M (2001) Crystallization screens: compatibility with the lipidic cubic phase for in meso crystallization of membrane proteins. Biophys J 81(1):225–242

    Article  Google Scholar 

  • Cherezov V, Peddi A, Muthusubramaniam L, Zheng YF, Caffrey M (2004) A robotic system for crystallizing membrane and soluble proteins in lipidic mesophases. Acta Crystallogr D 60:1795–1807

    Article  Google Scholar 

  • Cherezov V, Clogston J, Papiz MZ, Caffrey M (2006) Room to move: crystallizing membrane proteins in swollen lipidic mesophases. J Mol Biol 357(5):1605–1618

    Article  Google Scholar 

  • Cherezov V, Rosenbaum DM, Hanson MA, Rasmussen SGF, Thian FS, Kobilka TS, Choi HJ, Kuhn P, Weis WI, Kobilka BK, Stevens RC (2007) High-resolution crystal structure of an engineered human beta(2)-adrenergic G protein-coupled receptor. Science 318(5854):1258–1265

    Article  Google Scholar 

  • Cline SW, Doolittle WF (1987) Efficient transfection of the archaebacterium Halobacterium halobium. J Bacteriol 169(3):1341–1344

    Google Scholar 

  • Davis J, Donohue TJ, Kaplan S (1988) Construction, characterization, and complementation of a Puf- mutant of Rhodobacter sphaeroides. J Bacteriol 170(1):320–329

    Google Scholar 

  • Duffy DC, McDonald JC, Schueller OJA, Whitesides GM (1998) Rapid prototyping of microfluidic systems in poly(dimethylsiloxane). Anal Chem 70(23):4974–4984

    Article  Google Scholar 

  • Jaakola VP, Griffith MT, Hanson MA, Cherezov V, Chien EYT, Lane JR, Ijzerman AP, Stevens RC (2008) The 2.6 angstrom crystal structure of a human A(2A) adenosine receptor bound to an antagonist. Science 322(5905):1211–1217

    Article  Google Scholar 

  • Katona G, Andreasson U, Landau EM, Andreasson LE, Neutze R (2003) Lipidic cubic phase crystal structure of the photosynthetic reaction centre from Rhodobacter sphaeroides at 2.35 angstrom resolution. J Mol Biol 331(3):681–692

    Article  Google Scholar 

  • Lau BTC, Baitz CA, Dong XP, Hansen CL (2007) A complete microfluidic screening platform for rational protein crystallization. J Am Chem Soc 129(3):454–455

    Article  Google Scholar 

  • Li L, Mustafi D, Fu Q, Tereshko V, Chen DLL, Tice JD, Ismagilov RF (2006) Nanoliter microfluidic hybrid method for simultaneous screening and optimization validated with crystallization of membrane proteins. Proc Natl Acad Sci USA 103(51):19243–19248

    Article  Google Scholar 

  • McDonald JC, Duffy DC, Anderson JR, Chiu DT, Wu HK, Schueller OJA, Whitesides GM (2000) Fabrication of microfluidic systems in poly(dimethylsiloxane). Electrophoresis 21(1):27–40

    Article  Google Scholar 

  • Nollert P (2002) From test tube to plate: a simple procedure for the rapid preparation of microcrystallization experiments using the cubic phase method. J Appl Crystallogr 35:637–640

    Article  Google Scholar 

  • Nollert P (2004) Lipidic cubic phases as matrices for membrane protein crystallization. Methods 34(3):348–353

    Article  Google Scholar 

  • Ostermeier C, Michel H (1997) Crystallization of membrane proteins. Curr Opin Struct Biol 7(5):697–701

    Article  Google Scholar 

  • Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. In: Carter CW, Sweet RM (eds) Macromolecular crystallography, Pt A, vol 276, Academic Press Inc., San Diego, pp 307–326

  • Peddi A, Muthusubramaniam L, Zheng YR, Cherezov V, Misquitta Y, Caffrey M (2007) High-throughput automated system for crystallizing membrane proteins in lipidic mesophases. IEEE Trans Autom Sci Eng 4(2):129–140

    Article  Google Scholar 

  • Perry SL, Roberts GW, Tice JD, Gennis RB, Kenis PJA (2009) Microfluidic generation of lipidic mesophases for membrane protein crystallization. Cryst Growth Des 9(6):2566–2569

    Article  Google Scholar 

  • Pokkuluri PR, Laible PD, Deng YL, Wong TN, Hanson DK, Schiffer M (2002) The structure of a mutant photosynthetic reaction center shows unexpected changes in main chain orientations and quinone position. Biochemistry 41(19):5998–6007

    Article  Google Scholar 

  • Roach LS, Song H, Ismagilov RF (2005) Controlling nonspecific protein adsorption in a plug-based microfluidic system by controlling interfacial chemistry using fluorous-phase surfactants. Anal Chem 77(3):785–796

    Article  Google Scholar 

  • Shestopalov I, Tice JD, Ismagilov RF (2004) Multi-step synthesis of nanoparticles performed on millisecond time scale in a microfluidic droplet-based system. Lab Chip 4(4):316–321

    Article  Google Scholar 

  • Shim JU, Cristobal G, Link DR, Thorsen T, Jia YW, Piattelli K, Fraden S (2007) Control and measurement of the phase behavior of aqueous solutions using microfluidics. J Am Chem Soc 129(28):8825–8835

    Article  Google Scholar 

  • Simon R, Priefer U, Puhler A (1983) A broad host range mobilization system for invivo genetic-engineering: transposon mutagenesis in gram-negative bacteria. Bio-Technology 1(9):784–791

    Google Scholar 

  • Song H, Tice JD, Ismagilov RF (2003) A microfluidic system for controlling reaction networks in time. Angew Chem Int Ed 42(7):768–772

    Google Scholar 

  • Song H, Chen DL, Ismagilov RF (2006a) Reactions in droplets in microflulidic channels. Angew Chem Int Ed 45(44):7336–7356

    Article  Google Scholar 

  • Song H, Li HW, Munson MS, Van Ha TG, Ismagilov RF (2006b) On-chip titration of an anticoagulant argatroban and determination of the clotting time within whole blood or plasma using a plug-based microfluidic system. Anal Chem 78(14):4839–4849

    Article  Google Scholar 

  • Taguchi AK W, Stocker JW, Alden RG, Causgrove TP, Peloquin JM, Boxer SG, Woodbury NW (1992) Biochemical-characterization and electron-transfer reactions of Sym1, a Rhodobacter capsulatus reaction center symmetry mutant which affects the initial electron-donor. Biochemistry 31(42):10345–10355

    Article  Google Scholar 

  • Theiler R, Suter F, Zuber H, Cogdell RJ (1984) A comparison of the primary structures of the 2 B800–850-apoproteins from wild-type Rhodopseudomonas sphaeroides strain 2.4.1 and a carotenoidless mutant strain R26.1. FEBS Lett 175(2):231–237

    Article  Google Scholar 

  • Tiede DM, Vazquez J, Cordova J, Marone PA (1996) Time-resolved electrochromism associated with the formation of quinone anions in the Rhodobacter sphaeroides R26 reaction center. Biochemistry 35(33):10763–10775

    Article  Google Scholar 

  • Toepke MW, Beebe DJ (2006) PDMS absorption of small molecules and consequences in microfluidic applications. Lab Chip 6(12):1484–1486

    Article  Google Scholar 

  • Wadsten P, Wohri AB, Snijder A, Katona G, Gardiner AT, Cogdell RJ, Neutze R, Engstrom S (2006) Lipidic sponge phase crystallization of membrane proteins. J Mol Biol 364(1):44–53

    Article  Google Scholar 

  • Wohri AB, Johansson LC, Wadsten-Hindrichsen P, Wahlgren WY, Fischer G, Horsefield R, Katona G, Nyblom M, Oberg F, Young G, Cogdell RJ, Fraser NJ, Engstrom S, Neutze R (2008) A lipidic-sponge phase screen for membrane protein crystallization. Structure 16(7):1003–1009

    Article  Google Scholar 

  • Zheng B, Roach LS, Ismagilov RF (2003) Screening of protein crystallization conditions on a microfluidic chip using nanoliter-size droplets. J Am Chem Soc 125(37):11170–11171

    Article  Google Scholar 

  • Zheng B, Gerdts CJ, Ismagilov RF (2005) Using nanoliter plugs in microfluidics to facilitate and understand protein crystallization. Curr Opin Struct Biol 15(5):548–555

    Article  Google Scholar 

Download references

Acknowledgments

This work was supported through Accelerated Technologies Center for Gene to 3D Structure (ATCG3D) funded by the National Institute of General Medical Sciences (NIGMS), National Center for Research Resources under the PSI-2 Specialized Center program (U54 GM074961); the National Institutes of Health Roadmap for Medical Research (R01 GM075827 and P01 GM75913), and University of Chicago/Argonne National Laboratory (ANL) Collaborative Seed Funding. We thank Nina Ponomarenko and James R. Norris at the University of Chicago for samples of Reaction Center from B. viridis. We thank Ray C. Stevens and Peter Kuhn for helpful discussion and Elizabeth B. Haney for contributions in writing and editing this manuscript. Use of the ANL General Medicine and Cancer Institute Collaborative Access Team (GM/CA CAT) beamlines at the Advanced Photon Source was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Science, under Contract No. DE-AC02-06CH11357. GM/CA CAT has been funded in whole or in part with Federal funds from the National Cancer Institute (Y1-CO-1020) and the NIGMS (Y1-GM-1104).

Author information

Affiliations

  1. Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, IL, 60637, USA

    Liang Li, Qiang Fu & Rustem F. Ismagilov

  2. Biosciences Division, Argonne National Laboratory, 9700 South Cass Ave., Argonne, IL, 60439, USA

    Christopher A. Kors & Philip D. Laible

  3. deCODE biostructures, Accelerated Technologies Center for Gene to 3D Structure, 7869 NE Day Rd. W, Bainbridge Island, WA, 98110, USA

    Lance Stewart

  4. Emerald BioSystems, Inc., 7869 NE Day Rd. W, Bainbridge Island, WA, 98110, USA

    Peter Nollert

Authors
  1. Liang Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Qiang Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christopher A. Kors
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lance Stewart
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Peter Nollert
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Philip D. Laible
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Rustem F. Ismagilov
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Rustem F. Ismagilov.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 20 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Li, L., Fu, Q., Kors, C.A. et al. A plug-based microfluidic system for dispensing lipidic cubic phase (LCP) material validated by crystallizing membrane proteins in lipidic mesophases. Microfluid Nanofluid 8, 789–798 (2010). https://doi.org/10.1007/s10404-009-0512-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10404-009-0512-8

Keywords

  • Droplet
  • Plugs
  • Lipidic cubic phase
  • Membrane protein
  • Protein crystallization