Overcoming barriers to membrane protein structure determination

Bill, Roslyn M.; Henderson, Peter J. F.; Iwata, So; Kunji, Edmund R. S.; Michel, Hartmut; Neutze, Richard; Newstead, Simon; Poolman, Berend; Tate, Christopher G.; Vogel, Horst

doi:10.1038/nbt.1833

Cited by 332 publications

(290 citation statements)

References 94 publications

(112 reference statements)

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“…Accordingly, in recent years much effort has been made to find expression systems that yield sufficient amounts of native-like protein and detergents/lipids that mimic the native membrane environment [3,4]. Nevertheless, IM proteins are still dramatically under-represented in the three-dimensional structural database of the Protein Data Bank (PDB; see http://www.pdb.org/ and [5]).…”

Section: Introductionmentioning

confidence: 99%

Expression and purification of integral membrane metallopeptidase HtpX

Arolas

García‐Castellanos

Goulas

et al. 2014

Protein Expression and Purification

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The authors state they have no competing financial interest Arolas et al. ! 2! Highlights• The integral membrane metallopeptidase HtpX was overexpressed in E. coli.• A catalytically ablated HtpX mutant was designed to avoid self-cleavage.• The protein was extracted from membranes and purified in octyl-!-D-glucoside.• The protein was purified by cobalt-affinity, anion-exchange and size-exclusion.• The overall system renders milligram amounts of HtpX and fosters structural studies. AbstractLittle is known about the catalytic mechanism of integral membrane (IM) peptidases. HtpX is an IM metallopeptidase that plays a central role in protein quality control by preventing the accumulation of misfolded proteins in the membrane. Here we report the recombinant overexpression and purification of a catalytically ablated form of HtpX from Escherichia coli.Several E. coli strains, expression vectors, detergents, and purification strategies were tested to achieve maximum yields of pure and well-folded protein. HtpX was successfully overexpressed in E. coli BL21(DE3) cells using a pET-derived vector attaching a C-terminal His 8 -tag, extracted from the membranes using octyl-!-D-glucoside, and purified to homogeneity in the presence of this detergent in three consecutive steps: cobalt-affinity, anion-exchange, and sizeexclusion chromatography. The production of HtpX in milligram amounts paves the way for structural studies, which will be essential to understand the catalytic mechanism of this IM peptidase and related family members.

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Section: Introductionmentioning

confidence: 99%

Expression and purification of integral membrane metallopeptidase HtpX

Arolas

García‐Castellanos

Goulas

et al. 2014

Protein Expression and Purification

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“…These advances are a result of many factors (Bill et al, 2011), including improvements in membrane protein overexpression, stabilization of proteins using antibodies or thermostabilizing mutations, and the enhancement of crystallization technologies such as crystallization in lipidic cubic phase (LCP, in meso crystallization). However, there are still many challenges associated with membrane protein crystallization, data collection and structure determination.…”

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confidence: 99%

Advances in membrane protein crystallography: in situ and in meso data collection

Weyand

Tate

2015

Acta Cryst D Biol Crystallogr

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Membrane protein structural biology has made tremendous advances over the last decade as indicated by the exponential growth in the number of structures that have been published (http://blanco.biomol.uci.edu/mpstruc/). These advances are a result of many factors (Bill et al., 2011), including improvements in membrane protein overexpression, stabilization of proteins using antibodies or thermostabilizing mutations, and the enhancement of crystallization technologies such as crystallization in lipidic cubic phase (LCP, in meso crystallization). However, there are still many challenges associated with membrane protein crystallization, data collection and structure determination. Major problems often arise because membrane proteins frequently form tiny crystals, which either cannot be improved in size or which can be improved in size, but, as a consequence, lose diffraction quality. In addition, crystal handling, such as mounting the crystals and soaking in cryoprotectants, is often the reason for the loss of diffraction quality through mechanical shear-induced microlesions. This is particularly true for membrane protein crystals, which are often very fragile because of their high solvent content and being very thin in one dimension. In this issue of Acta Cryst. D, two independent groups, Axford et al. (2015) and Huang et al. (2015), have published methods that make a major contribution to addressing these problems, which will facilitate high-resolution data-collection of fragile crystals.In the methodology demonstrated by Axford et al. (2015), a standard in situ 96-well sitting-drop crystallization plate was used to crystallize TehA from Haemophilus influenzae in a final volume of 200 nl. The plate was left for several days until crystals grew to their maximum size (up to 75 mm in the largest dimension). Instead of harvesting and mounting the crystals, the team mounted the entire plate on the beamline (in this case I24 at the Diamond Light Source; Fig. 1) and standard procedures were then used for data collection from the membrane protein crystals, i.e. each crystal was centered in the beam and wedges of data were collected at room temperature. This avoided simultaneously two major potential problems, namely crystal handling and cryocooling. Wedges of data were collected from multiple different crystals (30-50 images per wedge, 0.2 rotation each), thus reducing the effects of radiation damage, and then they were merged to obtain a data set at 2.3 Å resolution (90% complete). A direct comparison was performed between the structure of TehA determined from a cryocooled crystal at 1.5 Å resolution (98% complete, one crystal) and from the room temperature plate collection strategy (56 crystals); only minor changes were observed in flexible regions such as loop regions. This study therefore provides a proof of principle that membrane protein structures can be determined at a synchrotron using in situ room temperature data collection strategies. Huang et al. (2015) took the in situ approach one step further and showed the...

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“…In recent years, several approaches to stabilize membrane proteins have been explored (9). These methods include development and optimization of solubilizing detergents (10), reconstitution of proteins into lipidic bicelles (11,12), cleavage of flexible protein regions (13)(14)(15)(16)(17)(18), co-crystallization with stabilizing ligands, antibodies, and fusion with soluble proteins (e.g., T4 lysozyme) (13-15, 17, 19-23).…”

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confidence: 99%

Naturally evolved G protein-coupled receptors adopt metastable conformations

Chen

Zhou

Fryszczyn

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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A wide range of membrane receptors signal through conformational changes, and the resulting protein conformational flexibility often hinders their structural studies. Because the determinants of membrane receptor conformational stability are still poorly understood, identifying a minimal set of perturbations stabilizing a membrane protein in a given conformation remains a major challenge in membrane protein structure determination. We present a novel approach integrating bioinformatics, computational design and experimental techniques that identifies and stabilizes metastable receptor regions. When applied to the beta1-adrenergic receptor, the method generated 13 novel receptor variants stabilized in the intended inactive state among which two exhibit an apparent thermostability higher than WT and M23 (a receptor variant previously stabilized by extensive scanning mutagenesis) by more than 30°C and 11°C, respectively. Targeted regions involve nonconserved unsatisfied polar residues or exhibit significant packing defects, features found in all class A G protein-coupled receptor structures. These findings suggest that natural G protein-coupled receptor sequences have evolved to be conformationally metastable through the design of suboptimal polar and van der Waals tertiary interactions. Given sufficiently accurate structural models, our approach should prove useful for designing stabilized variants of many uncharacterized membrane receptors.computational protein design | protein conformational stability | membrane protein modeling | signal transduction | synthetic biology M embrane proteins represent around 30% of currently sequenced genomes and are critical in the regulation of cell signaling and cell-cell communication (1, 2). When dysfunctional, however, these proteins can be responsible for serious diseases and constitute more than 60% of current drug targets. Despite their abundance and functional importance, membrane proteins are largely underrepresented in the Protein Data Bank, mainly due to technical difficulties in studying these proteins. A large fraction of these proteins such as G protein-coupled receptors (GPCRs) transduces signals across membranes through conformational changes (3-6). The resulting protein conformational heterogeneity and flexibility is a major factor hindering crystallization. An additional obstacle to structural determination is the poor stability of membrane proteins in detergent solution required by traditional crystallization approaches (7,8).In recent years, several approaches to stabilize membrane proteins have been explored (9). These methods include development and optimization of solubilizing detergents (10), reconstitution of proteins into lipidic bicelles (11, 12), cleavage of flexible protein regions (13-18), co-crystallization with stabilizing ligands, antibodies, and fusion with soluble proteins (e.g., T4 lysozyme) (13-15, 17, 19-23). Several of these modifications, however, disrupt important functional regions and interactions with either natural ligands or downstre...

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Overcoming barriers to membrane protein structure determination

Cited by 332 publications

References 94 publications

Expression and purification of integral membrane metallopeptidase HtpX

Expression and purification of integral membrane metallopeptidase HtpX

Advances in membrane protein crystallography: in situ and in meso data collection

Naturally evolved G protein-coupled receptors adopt metastable conformations

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