Detergents destabilize the cubic phase of monoolein: implications for membrane protein crystallization

The in meso method for membrane protein crystallization uses a lipidic cubic phase as the hosting medium. The cubic phase provides a lipid bilayer into which the protein presumably reconstitutes and from which protein crystals nucleate and grow. The solutions used to spontaneously form the protein-enriched cubic phase often contain significant amounts of detergents that were employed initially to purify and to solubilize the membrane protein. By virtue of their surface activity, detergents have the potential to impact on the phase properties of the in meso system and, by extension, the outcome of the crystallization process. The purpose of this study was to quantify the effects that a popular series of nonionic detergents, the n-alkyl-beta-D-glucopyranosides, have on the phase behavior of hydrated monoolein, the lipid upon which the in meso method is based. Phase identity and phase microstructure were characterized by small-angle x-ray diffraction on samples prepared to mimic in meso crystallization conditions. Measurements were made in the 0-40 degrees C range. Samples prepared in the cooling direction allow for the expression of metastability, a feature of liquid crystalline phases that might be exploited in low-temperature crystallization. The results show that the cubic phase is relatively insensitive to small amounts of alkyl glucosides. However, at higher levels the detergents trigger a transition to the lamellar phase in a temperature- and salt concentration-dependent manner. These effects have important implications for in meso crystallization. A diffraction-based method for assaying detergents is presented.     

Tricks of the trade used to accelerate high-resolution structure determination of membrane proteins

The rate at which X-ray structures of membrane proteins are solved is on a par with that of soluble proteins in the late 1970s. There are still many obstacles facing the membrane protein structural community. Recently, there have been several technical achievements in the field that have started to dramatically accelerate structural studies. Here, we summarize these so-called ‘tricks-of-the-trade’ and include case studies of several mammalian transporters.     

The transmembrane domain provides nucleotide binding specificity to the bacterial conjugation protein TrwB

In order to understand the functional significance of the transmembrane domain of TrwB, an integral membrane protein involved in bacterial conjugation, the protein was purified in the native, and also as a truncated soluble form (TrwBΔN70). The intact protein (TrwB) binds preferentially purine over pyrimidine nucleotides, NTPs over NDPs, and ribo- over deoxyribonucleotides. In contrast, TrwBΔN70 binds uniformly all tested nucleotides. The transmembrane domain has the general effect of making the nucleotide binding site(s) less accessible, but more selective. This is in contrast to other membrane proteins in which most of the protein mass, including the catalytic domain, is outside the membrane, but whose activity is not modified by the presence or absence of the transmembrane segment.     

Using micellar mole fractions to assess membrane protein stability in mixed micelles

The increased focus on the structural and physical properties of membrane proteins has made it critical to develop methods that provide a reliable estimate of membrane protein stability. A simple approach is to monitor the protein's conformational changes in mixed detergent systems, typically consisting of an anionic (denaturing) and non-ionic (non-denaturing) component. Linear correlations between, e.g., the melting temperature and the bulk mole fraction of the anionic component have been observed. However, a potential complication is that the bulk mole fraction is not identical to the mole fraction in the mixed micelle, which is the local environment experienced by the membrane protein. Here, we present an extensive analysis of the thermal stability of the membrane-integrated domain of the outer membrane protein AIDA in the presence of different mixed micelles. In the micelle system SDS-octyl-polyoxyethylene, the melting temperature in the absence of SDS extrapolates to 113 °C using bulk mole fractions. However, for mixed micelles involving short-chain detergents or phospholipids, the melting temperature calculated using bulk mole fractions reaches values up to several hundred degrees higher than 113 °C and can only be obtained by extrapolation over a narrow mole fraction interval. Furthermore, there is a non-linear relationship between the melting temperature and bulk mole fractions for mixed micelle systems involving cationic detergents (also denaturing). We show that if we instead use the micellar mole fraction as a parameter for denaturing detergent strength, we obtain linear correlations which extrapolate to more or less the same value of the melting temperature. There remains some scatter in the extrapolated values of the melting temperature in different binary systems, which suggest that additional micellar interactions may play a role. Nevertheless, in general terms, the mixed micellar composition is a good parameter to describe the membrane protein's microenvironment. Note, however, that for the mixed micelle system involving SDS and dodecyl maltoside, which has been used by several research groups to determine membrane protein stability, the estimate provided by bulk mole fraction leads to similar values as that of micellar mole fractions.     

Selecting Optimum Eukaryotic Integral Membrane Proteins for Structure Determination by Rapid Expression and Solubilization Screening

A medium-throughput approach is used to rapidly identify membrane proteins from a eukaryotic organism that are most amenable to expression in amounts and quality adequate to support structure determination. The goal was to expand knowledge of new membrane protein structures based on proteome-wide coverage. In the first phase, membrane proteins from the budding yeast Saccharomyces cerevisiae were selected for homologous expression in S. cerevisiae, a system that can be adapted to expression of membrane proteins from other eukaryotes. We performed medium-scale expression and solubilization tests on 351 rationally selected membrane proteins from S. cerevisiae. These targets are inclusive of all annotated and unannotated membrane protein families within the organism's membrane proteome. Two hundred seventy-two targets were expressed, and of these, 234 solubilized in the detergent n-dodecyl-β-d-maltopyranoside. Furthermore, we report the identity of a subset of targets that were purified to homogeneity to facilitate structure determinations. The extensibility of this approach is demonstrated with the expression of 10 human integral membrane proteins from the solute carrier superfamily. This discovery-oriented pipeline provides an efficient way to select proteins from particular membrane protein classes, families, or organisms that may be more suited to structure analysis than others.     

The Affinity Grid: a pre-fabricated EM grid for monolayer purification

We have recently developed monolayer purification as a rapid and convenient technique to produce specimens of His-tagged proteins or macromolecular complexes for single-particle electron microscopy (EM) without biochemical purification. Here, we introduce the Affinity Grid, a pre-fabricated EM grid featuring a dried lipid monolayer that contains Ni-NTA lipids (lipids functionalized with a nickel-nitrilotriacetic acid group). The Affinity Grid, which can be stored for several months under ambient conditions, further simplifies and extends the use of monolayer purification. After characterizing the Affinity Grid, we used it to isolate, within minutes, ribosomal complexes from Escherichia coli cell extracts containing His-tagged rpl3, the human homolog of the E. coli 50 S subunit rplC. Ribosomal complexes with or without associated mRNA could be prepared depending on the way the sample was applied to the Affinity Grid . Vitrified Affinity Grid specimens could be used to calculate three-dimensional reconstructions of the 50 S ribosomal subunit as well as the 70 S ribosome and 30 S ribosomal subunit from images of the same sample. We established that Affinity Grids are stable for some time in the presence of glycerol and detergents, which allowed us to isolate His-tagged aquaporin-9 (AQP9) from detergent-solubilized membrane fractions of Sf9 insect cells. The Affinity Grid can thus be used to prepare single-particle EM specimens of soluble complexes and membrane proteins.     

Membrane-permeabilizing motif in Semliki forest virus E1 glycoprotein

Cell infection by alphaviruses is accompanied by membrane permeability changes. New predictive approaches, including the computation of interfacial affinity and corresponding hydrophobic moments, suggest a segmented amphipathic-at-interface domain in the stem region of Semliki Forest virus fusion protein E1. Expression of E1 sequences in Escherichia coli cells confirmed that the membrane proximal plus transmembrane (TM) domain unit permeabilizes cells as efficiently as the 6K viroporin. Both our predictive and experimental data support the involvement of the E1 stem-TM region in membrane insertion and permeabilization. We propose to combine Wimley-White hydrophobicity analysis with expression-coupled permeability assays in order to identify viral products implied in breaching cell membrane barriers during infection.     

The funnel approach to the precrystallization production of membrane proteins

Challenges in the production of integral membrane proteins for structural studies include low expression levels, incorrect membrane insertion, aggregation and instability. In this report, we describe a “funnel approach” to overcoming these difficulties and demonstrate its efficacy in a case study of 36 prokaryotic P-type transporters. A diverse ensemble of modified constructs is generated and tested for expression in Escherichia coli, membrane localization, detergent extraction, and homogeneity. High-throughput methodologies are implemented throughout the process to facilitate identification of promising targets. We find that the choice of promoter, the choice of source organism providing the cloned gene, and, most importantly, the position of the affinity tag have a great effect on successful production. The latter had pronounced effects at all tested levels, from expression levels observed in whole cells to the extent of membrane insertion, and even on protein function. Following the initial streamlined screening, we were able to fine-tune and produce 9 of the 36 targets as materials suitable for crystallization or other structural studies.     

Studying subunit-subunit interactions in a bacterial ABC transporterby in vitro assembly

The thermostable arginine ABC transporter of Geobacillus stearothermophilus consists of a solute binding protein, ArtJ; a transmembrane subunit, ArtM; and a nucleotide-binding subunit, ArtP. An ArtM/His₆-ArtP complex was functionally assembled from separately purified subunits as demonstrated by assaying stimulation of its ATPase activity by arginine-loaded ArtJ in proteoliposomes. Studying in vitro assembly with variants carrying mutations in the conserved Q loop and/or the EAA loop of ArtP and ArtM, respectively, confirmed the predicted roles of both motifs in intersubunit signaling and physical interaction, respectively. In vitro assembly is considered a useful tool for investigating assembly defects of ABC transporters caused by mutations.     

New tensio-active molecules stabilize a human G protein-coupled receptor in solution

Structural characterization of membrane proteins is hampered by the instability of the isolated proteins in detergent solutions. Here, we describe a new class of phospholipid-like surfactants that stabilize the G protein-coupled receptor, BLT1. These compounds, called C(13)U(9), C(13)U(19), C(15)U(25) and C(17)U(16), were synthesized by radical polymerization of Tris(hydroxymethyl) acrylamidomethane in the presence of thioglycerol, first endowed with two hydrocarbon chains with variable lengths (13-17 carbon atoms), as transfer reagent. C(13)U(19), C(17)U(16) or C(15)U(25) significantly enhanced the stability of BLT1 in solution compared to what was obtained with common detergents. These molecules therefore represent a promising step towards the structural characterization of BLT1 and possibly other membrane proteins.     

Crucial steps in the structure determination of the Na+/H+ antiporter NhaA in its native conformation

Sodium proton antiporters are ubiquitous membrane proteins. Their importance for cell viability is the result of their role in homeostasis of intracellular pH, cellular Na+ content and cell volume. Recently, the first structure of this family of secondary transporters, namely of NhaA from Escherichia coli, revealed a novel fold and elucidated the molecular basis for the mechanism of transport and its regulation by pH. Here, we describe the key steps for the structure determination of NhaA, an iterative process of improving protein quality as well as crystallization conditions. Protein quality was optimized by shortening the purification to a single step and by changing the expression host. The major steps for crystal improvement were the exchange of the detergent during protein purification from the beta- to the alpha-anomer of DDM, the addition of OG to the crystallization set ups, and the growth of the crystals under conditions suitable for cryo-temperatures. Unexpectedly, the dimeric association of the transporter in the 3D crystal lattice is non-physiological. A comparison of the X-ray structure with the electron density map from cryo-electron microscopy of 2D crystals demonstrates that the NhaA helix packing in the 3D crystal is identical with the one in the lipid environment. Thus, the antiporter is in a native conformation in the 3D crystals.     

Optimizing Membrane Protein Overexpression in the Escherichia coli strain Lemo21(DE3)

Escherichia coli BL21(DE3) is widely used to overexpress proteins. In this overexpression host, the gene encoding the target protein is located on a plasmid and is under control of the T7 promoter, which is recognized exclusively by the T7 RNA polymerase (RNAP). The T7 RNAP gene is localized on the chromosome, and its expression is governed by the non-titratable, IPTG-inducible lacUV5 promoter. Recently, we constructed the Lemo21(DE3) strain, which allows improved control over the expression of genes from the T7 promoter. Lemo21(DE3) is a BL21(DE3) strain equipped with a plasmid harboring the gene encoding T7 lysozyme, an inhibitor of the T7 RNAP, under control of the exceptionally well-titratable rhamnose promoter. The overexpression yields of a large collection of membrane proteins in Lemo21(DE3) at different concentrations of rhamnose indicated that this strain may be very suitable for optimizing the production of membrane proteins. However, insight in the mechanism by which optimized expression yields are achieved in Lemo21(DE3) is lacking. Furthermore, whether the overexpressed proteins are suitable for functional and structural studies remains to be tested. Here, we show that in Lemo21(DE3), (i) the modulation of the activity of the T7 RNAP by the T7 lysozyme is key to optimizing the ratio of membrane proteins properly inserted in the cytoplasmic membrane to non-inserted proteins; (ii) maximizing the yields of membrane proteins is accompanied by reduction of the adverse effects of membrane protein overexpression, resulting in stable overexpression; and (iii) produced membrane proteins can be used for functional and structural studies.     

Solution NMR of signal peptidase, a membrane protein

Useful solution nuclear magnetic resonance (NMR) data can be obtained from full-length, enzymatically active type I signal peptidase (SPase I), an integral membrane protein, in detergent micelles. Signal peptidase has two transmembrane segments, a short cytoplasmic loop, and a 27-kD C-terminal catalytic domain. It is a critical component of protein transport systems, recognizing and cleaving amino-terminal signal peptides from preproteins during the final stage of their export. Its structure and interactions with the substrate are of considerable interest, but no three-dimensional structure of the whole protein has been reported. The structural analysis of intact membrane proteins has been challenging and only recently has significant progress been achieved using NMR to determine membrane protein structure. Here we employ NMR spectroscopy to study the structure of the full-length SPase I in dodecylphosphocholine detergent micelles. HSQC-TROSY spectra showed resonances corresponding to approximately 3/4 of the 324 residues in the protein. Some sequential assignments were obtained from the 3D HNCACB, 3D HNCA, and 3D HN(CO) TROSY spectra of uniformly ²H, ¹³C, ¹⁵N-labeled full-length SPase I. The assigned residues suggest that the observed spectrum is dominated by resonances arising from extramembraneous portions of the protein and that the transmembrane domain is largely absent from the spectra. Our work elucidates some of the challenges of solution NMR of large membrane proteins in detergent micelles as well as the future promise of these kinds of studies.     

A short helix in the C-terminal region of LolA is important for the specific membrane localization of lipoproteins

The structures of a lipoprotein carrier, LolA, and a lipoprotein receptor, LolB, are similar except for an extra C-terminal loop containing a 3(10) helix and beta-strand 12 in LolA. Lipoprotein release was significantly reduced when beta-12 was deleted. Deletion of the 3(10) helix also inhibited the lipoprotein release. Furthermore, lipoproteins were non-specifically localized to membranes when LolA lacked the 3(10) helix. Thus, the membrane localization of lipoproteins with the LolA derivative lacking the 3(10) helix was independent of LolB whereas LolB was essential for the outer membrane localization of lipoproteins with the wild-type LolA.     

Assembly of a transmembrane b-Type cytochrome is mainly driven by transmembrane helix interactions

Folding, assembly and stability of α-helical membrane proteins is still not very well understood. Several of these membrane proteins contain cofactors, which are essential for their function and which can be involved in protein assembly and/or stabilization. The effect of heme binding on the assembly and stability of the transmembrane b-type cytochrome b₅₅₉^′ was studied by fluorescence resonance energy transfer. Cytochrome b₅₅₉^′ consists of two monomers of a 44 amino acid long polypeptide, which contains one transmembrane domain. The synthesis of two variants of the b₅₅₉^′ monomer, each carrying a specific fluorescent dye, allowed monitoring helix-helix interactions in micelles by resonance energy transfer. The measurements demonstrate that the transmembrane peptides dimerize in detergent in the absence and presence of the heme cofactor. Cofactor binding only marginally enhances dimerization and, apparently, the redox state of the heme group has no effect on dimerization.     

Dephosphorylation of photosystem II proteins and phosphorylation of CP29 in barley photosynthetic membranes as a response to water stress

Kinetic studies of protein dephosphorylation in barley thylakoid membranes revealed accelerated dephosphorylation of photosystem II (PSII) proteins, and meanwhile rapidly induced phosphorylation of a light-harvesting complex (LHCII) b4, CP29 under water stress. Inhibition of dephosphorylation aggravates stress damages and hampers photosystem recovery after rewatering. This increased dephosphorylation is catalyzed by both intrinsic and extrinsic membrane protein phosphatase. Water stress did not cause any thylakoid destacking, and the lateral migration from granum membranes to stroma-exposed lamellae was only found to CP29, but not other PSII proteins. Activation of plastid proteases and release of TLP40, an inhibitor of the membrane phosphatases, were also enhanced during water stress. Phosphorylation of CP29 may facilitate disassociation of LHCII from PSII complex, disassembly of the LHCII trimer and its subsequent degradation, while general dephosphorylation of PSII proteins may be involved in repair cycle of PSII proteins and stress-response-signaling.     

Predicted bacteriorhodopsin from Exiguobacterium sibiricum is a functional proton pump

The predicted Exigobacterium sibiricum bacterirhodopsin gene was amplified from an ancient Siberian permafrost sample. The protein bacteriorhodopsin from Exiguobacterium sibiricum (ESR) encoded by this gene was expressed in Escherichia coli membrane. ESR bound all-trans-retinal and displayed an absorbance maximum at 534 nm without dark adaptation. The ESR photocycle is characterized by fast formation of an M intermediate and the presence of a significant amount of an O intermediate. Proteoliposomes with ESR incorporated transport protons in an outward direction leading to medium acidification. Proton uptake at the cytoplasmic surface of these organelles precedes proton release and coincides with M decay/O rise of the ESR.     

Two novel oligosaccharides isolated from a beverage produced by fermentation of a plant extract

An extract from 50 kinds of fruits and vegetables was fermented to produce a new beverage. Natural fermentation of the extract was carried out mainly by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp. and Pichia spp.). Two new saccharides were found in this fermented beverage. The saccharides were isolated using carbon-Celite column chromatography and preparative high performance liquid chromatography. Gas liquid chromatography analysis of methylated derivatives as well as MALDI-TOF MS and NMR measurements were used for structural confirmation. The (1)H and (13)C NMR signals of each saccharide were assigned using 2D-NMR including COSY, HSQC, HSQC-TOCSY, CH(2)-HSQC-TOCSY, and CT-HMBC experiments. The saccharides were identified as beta-D-fructopyranosyl-(2-->6)-beta-D-glucopyranosyl-(1-->3)-D-glucopyranose and beta-D-fructopyranosyl-(2-->6)-[beta-D-glucopyranosyl-(1-->3)]-D-glucopyranose.     

The cytochrome ba complex from the thermoacidophilic crenarchaeote Acidianus ambivalens is an analog of bc1 complexes

A novel cytochrome ba complex was isolated from aerobically grown cells of the thermoacidophilic archaeon Acidianus ambivalens. The complex was purified with two subunits, which are encoded by the cbsA and soxN genes. These genes are part of the pentacistronic cbsAB-soxLN-odsN locus. The spectroscopic characterization revealed the presence of three low-spin hemes, two of the b and one of the a_s-type with reduction potentials of + 200, + 400 and + 160 mV, respectively. The SoxN protein is proposed to harbor the heme b of lower reduction potential and the heme a_s, and CbsA the other heme b. The soxL gene encodes a Rieske protein, which was expressed in E. coli; its reduction potential was determined to be + 320 mV. Topology predictions showed that SoxN, CbsB and CbsA should contain 12, 9 and one transmembrane α-helices, respectively, with SoxN having a predicted fold very similar to those of the cytochromes b in bc₁ complexes. The presence of two quinol binding motifs was also predicted in SoxN. Based on these findings, we propose that the A. ambivalens cytochrome ba complex is analogous to the bc₁ complexes of bacteria and mitochondria, however with distinct subunits and heme types.     

Purification of the functional plant membrane channel KAT1

The inward-rectifying K⁺ channel KAT1 is expressed mainly in Arabidopsis thaliana guard cells. The purification of functional KAT1 has never been reported. We investigated the extraction of the plant K⁺ channel KAT1 with different detergents, as an example for how to select detergents for purifying a eukaryotic membrane protein. A KAT1-GFP fusion protein was used to screen a library of 46 detergents for the effective solubilization of intact KAT1. Then, a “test set” of three detergents was picked for further analysis, based on their biochemical characteristics and availability. The combination use of the selected detergents enabled the effective purification of functional KAT1 with affinity and gel-filtration chromatography.