December 2002
Volume 43, Issue 13
ARVO Annual Meeting Abstract  |   December 2002
Crystallisation of Lens Membrane Proteins for Structural Analysis
Author Affiliations & Notes
  • T Gonen
    School of Biological Sci Univ of Auckland Auckland New Zealand
  • Footnotes
    Commercial Relationships   T. Gonen, None.
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 4645. doi:
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      T Gonen; Crystallisation of Lens Membrane Proteins for Structural Analysis . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4645.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract: : Purpose: Approximately 20% of the genes in the human genome encode membrane-traversing proteins. Most structures determined to date are of soluble proteins. Structural studies of membrane proteins is hindered by the fact they comprise both hydrophilic and hydrophobic regions which make their handling, biochemistry and crystallisation a problem. Here we use two membrane proteins of the ocular lens, the water channel protein aquaporin 0 (AQP0), and a lens tetraspanin MP20, which is probably involved in cell-cell adhesion, to illustrate two different techniques for the structural analysis of membrane proteins: 3D crystallisation for X ray diffraction and 2D crystallisation for analysis by cryo-electron microscopy. Methods: Lens MIP and MP20 were detergent solubilised from fiber cell membranes and purified by FPLC. MIP was crystallised by the sitting drop method for analysis by X-ray crystallography while MP20 was reconstituted into artificial membranes to form highly ordered two-dimensional crystals for analysis by cryo electron microscopy. Result: MIP/AQP0 forms 3-dimensional crystals suitable for structural analysis. MP20 forms highly ordered tubes and was analysed by negative stain electron microscopy and packs as dimers in a P2 unit cell. Conclusion: Further work is needed to obtain the three dimensional structure of lens MIP and MP20. The enigmatic MP20 has no known function. It associates with galectin-3, which is an adhesion modulator, and is found in junctional domains. Future work will involve the three dimensional model of MP20 interacting with its partner galectin-3.

Keywords: 526 protein purification and characterization • 527 protein structure/function 

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