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Crystallization of Membrane Proteins
Integral membrane proteins play a crucial role in many cellular
functions, such as ion regulation and transport, molecular recognition and
response, and energy transduction, and are targets for many pharmacological
compounds (figure from Stryer, Biochemistry). Our understanding of the function
of this broad range of proteins is highly circumscribed by a lack of structural
information, due to difficulty in obtaining crystals suitable for X-ray diffraction.
(See also: Membrane proteins,
X-ray diffraction)
The limited success in crystallizing integral membrane proteins can be attributed to several factors. Integral membrane proteins are more difficult to isolate than water-soluble proteins, as the native membrane surrounding the protein must be disrupted and replaced with detergent molecules without causing any denaturation. (See: Lipids and Detergents)
The solubility of a protein is a critical parameter in the crystallization
process. As a result, dependences of solubility on many factors such as the
ionic strength and pH have been well-characterized for water soluble proteins.
In contrast, factors influencing the solubility of integral membrane proteins,
most notably the detergent, are poorly characterized. We have developed, a
relatively simple solubility assay that is applicable to structural investigations
of a variety of integral membrane proteins.
The crystal structure of bacterial reaction centers shows the
presence of lipid molecules on the surface of the protein (see Figure). The
presence
of these three lipids was confirmed by MALDI, matrix-assisted laser desorption
ionization. The lipids are located in the hydrophobic region of the protein
surface and interact predominately with hydrophobic amino acids, in particular
aromatic residues. The lipids span a distance of ~ 30 Å that is consistent
with a bilayer-like arrangement suggesting the presence of an "inner shell" of
lipids around membrane proteins that is critical for membrane function. The
identification of lipids in the structures of the bacterial reaction center
and other proteins suggests a critical role of lipids in determining the biochemical
properties of purified membrane proteins.
Related publications:
M. A. Rosenow, J. C. Williams, and J. P. Allen (2000) “Amphiphiles modify
the properties of detergent solutions used in crystallization of membrane proteins”,
Acta Crystallographica D 57, 925-927.
A. Camara-Artigas, C. L. Magee, J. C. Williams, and J. P. Allen (2001) “Individual interactions influence the crystalline order for membrane proteins” Acta Crystallographica D57, 1281-1286.
A. Camara-Artigas, D. Brune, and J. P. Allen (2002) “Interactions between lipids and bacterial reaction centers determined by protein crystallography” Proc. Natl. Acad. Sci. USA 99, 11055-11060. (PDF)
M. A. Rosenow, C. L. Magee, J. C. Williams, and J. P. Allen (2002) “The influence of detergents on the solubility of membrane proteins”, Acta Crystallographica D 58, 2076-2081. (PDF)
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13 February 2006 |
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