Crystallizing proteins on lipid monolayers

Jaap Brink, Ph.D.

W.K. Keck Center for Computational Biology and Verna and Marrs McLean Department for Biochemistry, Rm. 326B
Baylor College of Medicine, 1 Baylor Plaza, Houston TX 77030, U.S.A.

The most suitable specimen for electron crystallography is a two-dimensional (2D) crystal of a protein. Using the lipid monolayer technique, the protein of interest can be bound to the lipid layer via either specific or non-specific interactions. Thus, the protein's number of degrees of freedom have been reduced from 6 to 3. Also, the protein gets effectively concentrated at the lipid monolayer by up to 500 mg/ml (Kornberg and Darst). Thus, the protein is more likely to form 2D crystals. Using lipids doped with specific ligands unique orientations are easily obtained.
[Image of monolayer crystal] Crystals of proteins on monolayers of phospholipids are typically grown in small wells. Other methods of growing these crystals utilize Langmuir troughs. Generally, a small aliquot of protein solution is placed in such a well. A droplet with phospholipids is laid on top of this solution whereby it creates a monolayer. The protein shown here is the streptavidin schematically shown as red entities with 4 binding sites for biotin. The lipid is shown as a line of blue molecules with the biotin (in yellow) interspersed.
[Image showing modes of interaction] Crystallization of the protein can occur through special interactions between the protein (in red) and derivatized phospholipid with special recognition sites (in yellow). Shown is streptavidin bound to a layer of phosphatidylcholine (in blue) with derivatized biotin-containing lipid (in yellow). Examples of crystals grown using specific interaction are for instance, streptavidin, HIV reverse transcriptase and botulinum toxin. Alternatively, crystallization occurs through electrostatic interactions. Examples of such crystals are for instance ribosomes and RNA polymerase.
[Image detailing transfer step] Once the crystal has formed, it is transferred to a grid by simply putting the grid momentarily onto the well with the monolayer and crystallized protein. Different results on the transfer efficiency have been reported depending on the type of a grid, or whether a wire loop is used. After transfer, the grid can be frozen in liquid ethane thereby vitrifying the buffer, or the protein can be embedded in a thin layer of glucose.
Images courtesy of Dr. A. Avila-Sakar, BCM, 1995

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Created: May 1st 1996
Last update: July 12th 1996