Elsevier

Methods

Volume 34, Issue 3, November 2004, Pages 348-353
Methods

Lipidic cubic phases as matrices for membrane protein crystallization

https://doi.org/10.1016/j.ymeth.2004.03.030Get rights and content

Abstract

This review provides detailed procedures for the crystallization of membrane proteins via the lipidic cubic phase method. Bacteriorhodopsin-specific, hands-on protocols are given for (i) the preparation of bacteriohordopsin from purple membrane by monomerization in octylglucoside and gel filtration chromatography or by selective extraction after pre-treatment with dodecyl-trimethylammonium bromide, (ii) the incorporation of bacteriorhodopsin into lipidic cubic phases by mixing in vials or within coupled syringes and, (iii) the crystallization of bacteriorhodopsin in the lipidic matrix by adding a solid salt or an overlaying with a solution. References for further useful procedures and materials are listed in order to provide biochemists and crystallographers with all information that is necessary to grow crystals of the membrane protein bacteriorhodopsin.

Introduction

It is the purpose of this review to familiarize the experimental scientist with the practicalities involved in the use of lipidic cubic phases for membrane protein crystallization purposes. The detailed protocols given here enable the reader to readily generate crystals of the membrane protein bacteriorhodopsin. After mastering the crystallization of bacteriorhodopsin, further resources may be consulted, e.g., those listed in Table 1. It lists references that describe useful procedures and lipidic cubic phase related crystallization reports which may be instrumental in designing crystallization screens, handling of non-colored proteins, or the retrieval of crystals for X-ray diffraction purposes. It is hoped that the simple protocols in this review serve as a basis for benchmark crystallization experiments that embolden the crystallographer to experiment with more difficult proteins, in particular those that have not yielded crystals employing conventional crystallization methods.

The track record of the lipidic cubic phase crystallization method is summarized in Table 2. It has been particularly successful for membrane proteins with seven transmembrane α-helices (bacteriorhodopsin, halorhodopsin, sensory rhodopsin II, and sensory rhodopsin II with its transducer domain). It is therefore believed that heptahelical membrane proteins that are of non-bacterial origin, namely G-protein coupled receptors (GPCR), may be appropriate targets for this crystallization method. After its inception and description in 1996 [9], the crystallization of bacteriorhodopsin has been repeated in many laboratories (Table 2). The reproduction of well-diffracting bacteriorhodopsin crystals, the application of the method to other membrane proteins, and the continuing development of lipidic cubic phase-based crystallization technology demonstrate the value and future promise of this approach. Numerous original research reports and reviews have been published on the topic of lipidic cubic phase-based protein crystallization. For an introduction to this subject, consult Table 1.

Section snippets

Protocols: crystallization of bacteriorhodopsin in practice

In general, lipidic cubic phase-based crystallizations are often carried out as a two-step process. At first the protein solution is mixed with the lipid, and a lipidic cubic phase forms spontaneously. This first step is considered complete when the material is transparent, non-birefringent, and very viscous. Then the second step, crystallization, is started by the addition of a crystallant. The latter may be a solid salt mix or a solution. Depending on the precise conditions, crystals

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