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Liposomes pp 83-94 | Cite as

Liposomal Reconstitution of Monotopic Integral Membrane Proteins

  • Zahra MirAfzaliEmail author
  • David L. DeWitt
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 606)

Abstract

In spite of considerable progress in the methodology for reconstitution of membrane proteins into the liposomes, a successful reconstitution still appears to be more an art than a science. Reconstitution of membrane proteins into bilayers is required for establishing several aspects of the functions of membrane proteins and lipids and for elaborating models of naturally occurring membranes.

Cyclooxygenase (COX)-1 and -2 (also prostaglandin endoperoxide H2 synthase, PGHS-1 and -2) belong to the class of monotopic membrane proteins. Membrane-binding domains of both COX-1 and -2 contain four short, consecutive, amphipathic α-helices (A, B, C, and D). Crystal structures of the COXs indicate that basic, hydrophobic, and aromatic residues in the membrane-binding domain are oriented away from the protein core and form a surface on the enzyme, which has been proposed to interact with the lipid bilayer (1).

In this chapter, we describe a fast and efficient method for direct incorporation of COX-1 and -2 isozymes - as models for monotopic integral membrane proteins - into preformed liposomes containing fatty acids without loss of activity.

Key words

Monotopic membrane protein Proteoliposomes Liposome reconstitution Direct incorporation Membrane defect Incorporating impurities into membranes Cyclooxygenase enzyme 

Notes

Acknowledgments

The authors would like to thank Dr. Alicia Pastor and Mr. Robert Pcionek from Michigan State University Center for Advanced Microscopy for their help with the electron microscopy work.

References

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    Picot D, Loll PJ, Garavito RM (1994) The X-ray crystal structure of the membrane protein prostaglandin H2 synthase-1. Nature 367:243-249CrossRefPubMedGoogle Scholar
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    Jain MK, Zakim D (1987) The spontaneous incorporation of proteins into preformed bilayers. Biochim Biophys Acta 906:33-67PubMedGoogle Scholar
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    Smith T, Leipprandt J, DeWitt D (2000) Purification and characterization of the human recombinant histidine-tagged prostaglandin endo­­peroxide H synthases-1 and -2. Arch Biochem Biophys 375:195-200CrossRefPubMedGoogle Scholar
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    Mirafzali Z, Leipprandt J, DeWitt D (2005) Fast, efficient reconstitution of cyclooxygenases into proteoliposomes. Arch Biochem Biophys 443:60-65CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Encapsula NanoSciences LLCNashvilleUSA
  2. 2.Department of Biochemistry and Molecular BiologyMichigan State UniversityEast LansingUSA

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