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Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR) is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example. Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression. This complementary protein approach allowed us to increase bR expression by two orders of magnitude through the introduction of two silent mutations into bR coding DNA. For the first time the high quality crystals of bR expressed in E. Coli were obtained using the produced protein. The crystals obtained with in meso nanovolume crystallization diffracted to 1.67 Å. [ABSTRACT FROM AUTHOR]

Abstract: Here we report a successful use of a recently developed isoprenoid-chained lipid family for in meso crystallization of membrane proteins. The isoprenoid-chained lipid 1-O-(3,7,11,15-tetramethylhexadecyl)-β–d-xyloside (β-XylOC16+4) used as a host lipid for in meso crystallization provided high quality bacteriorhodopsin (bR) crystals (P63 space group) diffracting to high resolution and characterized by low twinning ratio. β-XylOC16+4 has an isoprenoid chain with methyl branches at each 4th position and a xylose group in the water-soluble part. These peculiarities make the lipid clearly distinguishable in the bR crystalline lattice and provides a unique opportunity to study the role of the host lipid in the in meso crystallization. We conclude that β-XylOC16+4 may have a general application for in meso crystallization for a wide range of membrane proteins. The cubic phase of β-XylOC16+4 is present over a wide range of temperatures and is stable at low temperature (down to about 8°C). This opens up the possibility of using temperature as a tool for the optimization of in meso crystallization with additional advantages for the crystallization of membrane proteins at lower temperatures where the proteins of interest may be more stable. [ABSTRACT FROM AUTHOR]

Twinning is one of the most common crystal-growth defects in protein crystallography. There are neither efficient rational approaches for the growth of nontwinned protein crystals nor are there examples of systematic studies of the dependence of the twinning-ratio distribution on crystallization conditions. The description of the twinning phenomenon has been covered even less for membrane-protein crystals and is nonexistent for crystals grown using lipidic phases (in meso). In the present work, possibilities for overcoming merohedral twinning are investigated for crystals of the membrane protein bacteriorhodopsin (bR) grown in meso. It is shown that traditional crystallization additives are not effective in the case of the in meso crystallization of bR. The twinning ratio was determined for 310 crystals grown under different crystallization conditions. A correlation of the twinning ratio with the growth rate of the crystals was observed. Slow growth indicated that crystals had a noticeable chance of avoiding twinning. Model calculations were performed in order to rationalize this observation. The calculations confirmed the experimental observation that most crystals consist of two twin domains and showed that under this condition small changes in the probability of twin-domain formation lead to dramatic changes in the number of nontwinned crystals, which explains why slow crystal growth results in a considerable number of nontwinned crystals. [ABSTRACT FROM AUTHOR]

The lipidic cubic phase (LCP) has been widely recognized as a promising membrane-mimicking matrix for biophysical studies of membrane proteins and their crystallization in a lipidic environment. Application of this material to a wide variety of membrane proteins, however, is hindered due to a limited number of available host lipids, mostly monoacylglycerols (MAGs). Here, we designed, synthesized, and characterized a series of chemically stable lipids resistant to hydrolysis, with properties complementary to the widely used MAGs. In order to assess their potential to serve as host lipids for crystallization, we characterized the phase properties and lattice parameters of mesophases made of two most promising lipids at a variety of different conditions by polarized light microscopy and small-angle X-ray scattering. Both lipids showed remarkable chemical stability and an extended LCP region in the phase diagram covering a wide range of temperatures down to 4 °C. One of these lipids has been used for crystallization and structure determination of a prototypical membrane protein bacteriorhodopsin at 4 and 20 °C. [ABSTRACT FROM AUTHOR]