Monoolein lipid phases as incorporation and enrichment materials for membrane protein crystallization.

The crystallization of membrane proteins in amphiphile-rich materials such as lipidic cubic phases is an established methodology in many structural biology laboratories. The standard procedure employed with this methodology requires the generation of a highly viscous lipidic material by mixing lipid, for instance monoolein, with a solution of the detergent solubilized membrane protein. This preparation is often carried out with specialized mixing tools that allow handling of the highly viscous materials while minimizing dead volume to save precious membrane protein sample. The processes that occur during the initial mixing of the lipid with the membrane protein are not well understood. Here we show that the formation of the lipidic phases and the incorporation of the membrane protein into such materials can be separated experimentally. Specifically, we have investigated the effect of different initial monoolein-based lipid phase states on the crystallization behavior of the colored photosynthetic reaction center from Rhodobacter sphaeroides. We find that the detergent solubilized photosynthetic reaction center spontaneously inserts into and concentrates in the lipid matrix without any mixing, and that the initial lipid material phase state is irrelevant for productive crystallization. A substantial in-situ enrichment of the membrane protein to concentration levels that are otherwise unobtainable occurs in a thin layer on the surface of the lipidic material. These results have important practical applications and hence we suggest a simplified protocol for membrane protein crystallization within amphiphile rich materials, eliminating any specialized mixing tools to prepare crystallization experiments within lipidic cubic phases. Furthermore, by virtue of sampling a membrane protein concentration gradient within a single crystallization experiment, this crystallization technique is more robust and increases the efficiency of identifying productive crystallization parameters. Finally, we provide a model that explains the incorporation of the membrane protein from solution into the lipid phase via a portal lamellar phase.

The Protein Maker: an automated system for high-throughput parallel purification.

The Protein Maker is an automated purification system developed by Emerald BioSystems for high-throughput parallel purification of proteins and antibodies. This instrument allows multiple load, wash and elution buffers to be used in parallel along independent lines for up to 24 individual samples. To demonstrate its utility, its use in the purification of five recombinant PB2 C-terminal domains from various subtypes of the influenza A virus is described. Three of these constructs crystallized and one diffracted X-rays to sufficient resolution for structure determination and deposition in the Protein Data Bank. Methods for screening lysis buffers for a cytochrome P450 from a pathogenic fungus prior to upscaling expression and purification are also described. The Protein Maker has become a valuable asset within the Seattle Structural Genomics Center for Infectious Disease (SSGCID) and hence is a potentially valuable tool for a variety of high-throughput protein-purification applications.

A Plug-Based Microfluidic System for Dispensing Lipidic Cubic Phase (LCP) Material Validated by Crystallizing Membrane Proteins in Lipidic Mesophases.

This paper presents a plug-based microfluidic system to dispense nanoliter-volume plugs of Lipidic Cubic Phase (LCP) material and subsequently merge the LCP plugs with aqueous plugs. This system was validated by crystallizing membrane proteins in lipidic mesophases, including LCP. This system allows for accurate dispensing of LCP material in nanoliter volumes, prevents inadvertent phase transitions that may occur due to dehydration by enclosing LCP in plugs, and is compatible with the traditional method of forming LCP material using a membrane protein sample, as shown by the successful crystallization of bacteriorhodopsin from Halobacterium salinarum. Conditions for the formation of LCP plugs were characterized and presented in a phase diagram. This system was also implemented using two different methods of introducing the membrane protein: 1) the traditional method of generating the LCP material using a membrane protein sample and 2) Post LCP-formation Incorporation (PLI), which involves making LCP material without protein, adding the membrane protein sample externally to the LCP material, and allowing the protein to diffuse into the LCP material or into other lipidic mesophases that may result from phase transitions. Crystals of bacterial photosynthetic reaction centers from Rhodobacter sphaeroides and Blastochloris viridis were obtained using PLI. The plug-based, LCP-assisted microfluidic system, combined with the PLI method for introducing membrane protein into LCP, should be useful for minimizing consumption of samples and broadening the screening of parameter space in membrane protein crystallization.

Nanovolume optimization of protein crystal growth using the microcapillary protein crystallization system.

The Microcapillary Protein Crystallization System (MPCS) is a microfluidic, plug-based crystallization technology that generates X-ray diffraction-ready protein crystals in nanolitre volumes. In this study, 28 out of 29 (93%) proteins crystallized by traditional vapor diffusion experiments were successfully crystallized by chemical gradient optimization experiments using the MPCS technology. In total, 90 out of 120 (75%) protein/precipitant combinations leading to initial crystal hits from vapor diffusion experiments were successfully crystallized using MPCS technology. Many of the resulting crystals produced high-quality X-ray diffraction data, and six novel protein structures that were derived from crystals harvested from MPCS CrystalCards are reported.

Effects of impurities on membrane-protein crystallization in different systems.

When starting a protein-crystallization project, scientists are faced with several unknowns. Amongst them are these questions: (i) is the purity of the starting material sufficient? and (ii) which type of crystallization experiment is the most promising to conduct? The difficulty in purifying active membrane-protein samples for crystallization trials and the high costs associated with producing such samples require an extremely pragmatic approach. Additionally, practical guidelines are needed to increase the efficiency of membrane-protein crystallization. In order to address these conundrums, the effects of commonly encountered impurities on various membrane-protein crystallization regimes have been investigated and it was found that the lipidic cubic phase (LCP) based crystallization methodology is more robust than crystallization in detergent environments using vapor diffusion or microbatch approaches in its ability to tolerate contamination in the forms of protein, lipid or other general membrane components. LCP-based crystallizations produced crystals of the photosynthetic reaction center (RC) of Rhodobacter sphaeroides from samples with substantial levels of residual impurities. Crystals were obtained with protein contamination levels of up to 50% and the addition of lipid material and membrane fragments to pure samples of RC had little effect on the number or on the quality of crystals obtained in LCP-based crystallization screens. If generally applicable, this tolerance for impurities may avoid the need for samples of ultrahigh purity when undertaking initial crystallization screening trials to determine preliminary crystallization conditions that can be optimized for a given target protein.

The plug-based nanovolume Microcapillary Protein Crystallization System (MPCS).

The Microcapillary Protein Crystallization System (MPCS) embodies a new semi-automated plug-based crystallization technology which enables nanolitre-volume screening of crystallization conditions in a plasticware format that allows crystals to be easily removed for traditional cryoprotection and X-ray diffraction data collection. Protein crystals grown in these plastic devices can be directly subjected to in situ X-ray diffraction studies. The MPCS integrates the formulation of crystallization cocktails with the preparation of the crystallization experiments. Within microfluidic Teflon tubing or the microfluidic circuitry of a plastic CrystalCard, approximately 10-20 nl volume droplets are generated, each representing a microbatch-style crystallization experiment with a different chemical composition. The entire protein sample is utilized in crystallization experiments. Sparse-matrix screening and chemical gradient screening can be combined in one comprehensive ;hybrid' crystallization trial. The technology lends itself well to optimization by high-granularity gradient screening using optimization reagents such as precipitation agents, ligands or cryoprotectants.

Membrane protein crystallization in amphiphile phases: practical and theoretical considerations.

Integral membrane proteins are amphiphilic molecules. In order to enable chromatographic purification and crystallization, a complementary amphiphilic microenvironment must be created and maintained. Various types of amphiphilic phases have been employed in crystallizations and intricate amphiphilic microenvironmental structures have resulted from these and are found inside membrane protein crystals. In this review the process of crystallization is put into the context of amphiphile phase transitions. Finally, practical factors are considered and a pragmatic way is suggested to pursue membrane protein crystallization trials.

Lipidic cubic phases as matrices for membrane protein crystallization.

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.

Protein interactions and membrane geometry.

The difficulty in growing crystals for x-ray diffraction analysis has hindered the determination of membrane protein structures. However, this is changing with the advent of a new method for growing high quality membrane protein crystals from the lipidic cubic phase. Although successful, the mechanism underlying this method has remained unclear. Here, we present a theoretical analysis of the process. We show that it is energetically favorable for proteins embedded in the highly curved cubic phase to cluster together in flattened regions of the membrane. This stabilizes the lamellar phase, permitting its outgrowth from the cubic phase. A kinetic barrier-crossing model is developed to determine the free energy barrier to crystallization from the time-dependent growth of protein clusters. Determining the values of key parameters provides both a rational basis for optimizing the experimental procedure for membrane proteins that have not yet been crystallized and insight into the analogous cubic to lamellar transitions in cells. We also discuss the implications of this mechanism for protein sorting at the exit sites of the Golgi and endoplasmic reticulum and the general stabilization of membrane structures.

Microscope detection options for colorless protein crystals grown in lipidic cubic phases.

The use of lipidic cubic phases as crystal nucleation and growth matrices is becoming popular and has yielded crystals of soluble and membrane proteins. So far, all of the membrane proteins crystallized by this method have been colored. This feature has facilitated the detection of the often encountered microcrystals in initial screening rounds. Indeed, small colorless protein crystals have poor optical contrast as a result of the small differences in refractive index of the protein crystal and the surrounding lipidic cubic phase. While a perfect preparation of a lipidic cubic phase is transparent and optically isotropic, in a crystallization setup it frequently disguises crystals due to cracks, inclusions, surface distortions and phase boundaries. Here, several specialized microscopic techniques and illumination conditions are compared and it is found that sufficient contrast is generated by cross polarization microscopy and by Hoffman modulation contrast microscopy for the detection of colorless protein crystals.

Control of the selectivity of the aquaporin water channel family by global orientational tuning.

Aquaporins are transmembrane channels found in cell membranes of all life forms. We examine their apparently paradoxical property, facilitation of efficient permeation of water while excluding protons, which is of critical importance to preserving the electrochemical potential across the cell membrane. We have determined the structure of the Escherichia coli aquaglyceroporin GlpF with bound water, in native (2.7 angstroms) and in W48F/F200T mutant (2.1 angstroms) forms, and carried out 12-nanosecond molecular dynamics simulations that define the spatial and temporal probability distribution and orientation of a single file of seven to nine water molecules inside the channel. Two conserved asparagines force a central water molecule to serve strictly as a hydrogen bond donor to its neighboring water molecules. Assisted by the electrostatic potential generated by two half-membrane spanning loops, this dictates opposite orientations of water molecules in the two halves of the channel, and thus prevents the formation of a "proton wire," while permitting rapid water diffusion. Both simulations and observations revealed a more regular distribution of channel water and an increased water permeability for the W48F/F200T mutant.

Early structural rearrangements in the photocycle of an integral membrane sensory receptor.

Sensory rhodopsins are the primary receptors of vision in animals and phototaxis in microorganisms. Light triggers the rapid isomerization of a buried retinal chromophore, which the protein both accommodates and amplifies into the larger structural rearrangements required for signaling. We trapped an early intermediate of the photocycle of sensory rhodopsin II from Natronobacterium pharaonis (pSRII) in 3D crystals and determined its X-ray structure to 2.3 A resolution. The observed structural rearrangements were localized near the retinal chromophore, with a key water molecule becoming disordered and the retinal's beta-ionone ring undergoing a prominent movement. Comparison with the early structural rearrangements of bacteriorhodopsin illustrates how modifications in the retinal binding pocket of pSRII allow subtle differences in the early relaxation of photoisomerized retinal.

Crystallization of membrane proteins in cubo.

Our understanding of lipidic cubic phases for the crystallization of membrane proteins has advanced greatly since the inception of the concept in 1996, and the method is becoming well accepted. Several protocols that allow the efficient screening of crystallization conditions and handling of crystals are presented. State-of-the art micro techniques allow a large number of crystallization conditions to be tested using very small amounts of protein, and diffraction quality crystals can be grown in larger volumes in glass vials. In cubo crystallization conditions differ from those employed for detergent-solubilized proteins. Variations comprise the type of lipid matrix, detergent, protein, salt, temperature, hydration, pH, and pressure. Commercially available screening kits may be applied in order to define lead conditions. Once obtained, crystals may be removed from the surrounding cubic phase mechanically, by enzymatic hydrolysis, or by detergent solubilization. We anticipate this set of protocols to be applied successfully to larger, less stable, and noncolored membrane proteins in order to obtain well-diffracting crystals of membrane proteins that have so far evaded crystallization in the detergent-solubilized state.