Lipidic cubic phases as matrices for membrane protein crystallization
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
References (24)
- et al.
Biophys. J.
(2001) - et al.
Methods Enzymol.
(1982) - et al.
Biophys. J.
(2003) - et al.
J. Mol. Biol.
(1999) - et al.
FEBS Lett.
(1999) - et al.
Methods Enzymol.
(2002) - et al.
BBA
(2000) - et al.
J. Phys. II France
(1997) - et al.
Chem. Phys. Lip.
(1998) - et al.
Acta Cryst. D
(2000)
J. Appl. Cryst.
Proc. Natl. Acad. Sci. USA
Cited by (32)
Boosting photoelectrochemical performance of GaN nanowall network photoanode with bacteriorhodopsin
2020, International Journal of Hydrogen EnergySurfactant bilayers maintain transmembrane protein activity
2014, Biophysical JournalCitation Excerpt :In 1996 the first reported crystallization of a membrane protein was that of bacteriorhodopsin in a lipidic cubic phase (6). Since then, numerous membrane protein structures crystallized in meso have been reported (2,5,7–16). In 2011 the number of membrane proteins structures determined by the in meso method and deposited in the Protein Data Bank (www.mpdb.tcd.ie) (17) stood at 79 (15), and it is increasing steadily (18–20).
Novel mixed-heteroatom macrocycles via templating: A new protocol
2013, Tetrahedron LettersAutomatic liquid handling for life science: A critical review of the current state of the art
2012, Journal of Laboratory AutomationCitation Excerpt :Automatic handling of highly viscous biomaterials at the nanoliter volumes is practically challenging, but it is an important task in biology research and the drug industry. Viscous materials can adhere to any tools they touch, and the droplet release takes longer.120,121 The OSU researchers performed a great deal of research to discover the control factors for automatic contact dispensing of highly viscous materials at the nanoliter scale.122,123
LCP-T<inf>m</inf>: An assay to measure and understand stability of membrane proteins in a membrane environment
2010, Biophysical JournalCitation Excerpt :Cultivation of Halobacterium salinarum (strain S9) was carried out as described previously (16,17). Wild-type bR was solubilized with 1.2% w/v n-octyl-β-D-glucoside (OG, Anatrace) using purple membranes isolated from H. salinarum following established protocols (16,18). The concentration of bR was determined by absorbance at 550 nm using the extinction coefficient ɛ550 ∼5.8 × 104 M−1 cm−1 (5).
In cubo crystallization of membrane proteins
2010, Advances in Planar Lipid Bilayers and LiposomesCitation Excerpt :Understanding the mechanism could help us tailoring the crystallization process thereby saving enormous amount of efforts required for multiparameter screening. Different groups including the research groups of M. Caffrey, P. Nollert, V. Cherezov, E. Landau, R. Templer, and S. Tanaka working in the area of membrane protein crystallization and LCPs have explored various physicochemical and mechanistic issues of this method [14,17,18,27,31,36,48,51,61,74,75,78,89–91,105,106,112,122,124,127,135,136,141,143,144,146,149,152,155–175], and they have proposed a mechanism [27,172,176] mainly on the basis of bacteriorhodopsin–monoolein system (bR-MO). The in cubo crystallization seems to occur in two stages: Stage-I, before the addition of salt and Stage-II, after the salt addition.