High-throughput analytical gel filtration screening of integral membrane proteins for structural studies

Löw, Christian; Moberg, Per; Quistgaard, E.M.; Hedrén, Marie; Guettou, Fatma; Frauenfeld, Jens; Haneskog, Lars; Nordlund, P.

doi:10.1016/j.bbagen.2013.02.001

Cited by 29 publications

(28 citation statements)

References 48 publications

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“…Furthermore, gel filtration of ligand-bound S1R purified from human leukemia cells showed that ligand binding activity was associated with a protein of ∼100 kDa (2). Many other membrane receptors adopt an oligomeric state (66–68), and recombinant expression often leads to a distribution of these states (69). For example, when human serotonin receptor 3A was overexpressed in E. coli , the protein was detected as a mixture of oligomers, and the biologically active pentamer represented only 7% of the total protein (70).…”

Section: Discussionmentioning

confidence: 99%

The Oligomeric States of the Purified Sigma-1 Receptor Are Stabilized by Ligands

Gromek

Suchy

Meddaugh

et al. 2014

Journal of Biological Chemistry

117

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Background: Sigma-1 receptor (S1R) is an integral membrane ligand-binding receptor.Results: Gel filtration chromatography revealed oligomeric states that are stabilized by ligand binding and destabilized by mutations in the GXXXG integral membrane dimerization domain.Conclusion: Purified S1R binds small molecule ligands as an oligomer but not as a monomer.Significance: The results provide new insight into the function of S1R with ligands and proteins partners.

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Section: Discussionmentioning

confidence: 99%

The Oligomeric States of the Purified Sigma-1 Receptor Are Stabilized by Ligands

Gromek

Suchy

Meddaugh

et al. 2014

Journal of Biological Chemistry

117

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“…Multiple constructs (based on secondary structure prediction) of the genes coding for both isoforms of the A-subunit ( PPP2R1A and PPP2R1B ) as well as for different isoforms of the PR72/B-subunit ( PPP2R3A , PPP2R3B and PPP2R3C ) were cloned into the pNIC28-Bsa4 vector carrying a N-terminal Histidine-tag followed by a Tobacco Etch Virus (TEV) cleavage site using ligation independent cloning [41] and screened for expression and crystallization using established protocols [42], [43]. All expression vector vectors possess a T7 promoter and terminator sequence.…”

Section: Methodsmentioning

confidence: 99%

Structural and Biochemical Characterization of Human PR70 in Isolation and in Complex with the Scaffolding Subunit of Protein Phosphatase 2A

et al. 2014

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Protein Phosphatase 2A (PP2A) is a major Ser/Thr phosphatase involved in the regulation of various cellular processes. PP2A assembles into diverse trimeric holoenzymes, which consist of a scaffolding (A) subunit, a catalytic (C) subunit and various regulatory (B) subunits. Here we report a 2.0 Å crystal structure of the free B’’/PR70 subunit and a SAXS model of an A/PR70 complex. The crystal structure of B’’/PR70 reveals a two domain elongated structure with two Ca2+ binding EF-hands. Furthermore, we have characterized the interaction of both binding partner and their calcium dependency using biophysical techniques. Ca2+ biophysical studies with Circular Dichroism showed that the two EF-hands display different affinities to Ca2+. In the absence of the catalytic C-subunit, the scaffolding A-subunit remains highly mobile and flexible even in the presence of the B’’/PR70 subunit as judged by SAXS. Isothermal Titration Calorimetry studies and SAXS data support that PR70 and the A-subunit have high affinity to each other. This study provides additional knowledge about the structural basis for the function of B’’ containing holoenzymes.

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“…The MS channels of small conductance (MscS) from the Archaea Thermoplasma volcanium (T1 and T2) were identified as prime candidates for structural studies using our recently established screening pipeline [47]. They are well expressed and yield a monodisperse gel filtration peak in various detergents.…”

Section: Resultsmentioning

confidence: 99%

“…Indeed, both MS channels (T1, T2) from the archaea Thermoplasma volcanium described here have been screened for over-expression as C-terminal GFP fusion constructs previously [55], but were discarded due to insufficient expression levels. However, following our recently described high-throughput approach for identification of IMP targets for structural studies [47], which is independent of fluorescent fusion partners, we readily identified both channels as prime targets for structural studies. High expression levels (> 5mg/L of culture) and homogenous channel preparations after purification were achieved with N-terminally tagged constructs only, indicating that C-terminal tags or fusion partners might interfere with the correct folding and oligomeric assembly of the channel.…”

Section: Discussionmentioning

confidence: 99%

Nanobody Mediated Crystallization of an Archeal Mechanosensitive Channel

et al. 2013

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Mechanosensitive channels (MS) are integral membrane proteins and allow bacteria to survive sudden changes in external osmolarity due to transient opening of their pores. The efflux of cytoplasmic osmolytes reduces the membrane tension and prevents membrane rupture. Therefore these channels serve as emergency valves when experiencing significant environmental stress. The preparation of high quality crystals of integral membrane proteins is a major bottleneck for structure determination by X-ray crystallography. Crystallization chaperones based on various protein scaffolds have emerged as promising tool to increase the crystallization probability of a selected target protein. So far archeal mechanosensitive channels of small conductance have resisted crystallization in our hands. To structurally analyse these channels, we selected nanobodies against an archeal MS channel after immunization of a llama with recombinant expressed, detergent solubilized and purified protein. Here we present the characterization of 23 different binders regarding their interaction with the channel protein using analytical gel filtration, western blotting and surface plasmon resonance. Selected nanobodies bound the target with affinities in the pico- to nanomolar range and some binders had a profound effect on the crystallization of the MS channel. Together with previous data we show that nanobodies are a versatile and valuable tool in structural biology by widening the crystallization space for highly challenging proteins, protein complexes and integral membrane proteins.

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High-throughput analytical gel filtration screening of integral membrane proteins for structural studies

Cited by 29 publications

References 48 publications

The Oligomeric States of the Purified Sigma-1 Receptor Are Stabilized by Ligands

The Oligomeric States of the Purified Sigma-1 Receptor Are Stabilized by Ligands

Structural and Biochemical Characterization of Human PR70 in Isolation and in Complex with the Scaffolding Subunit of Protein Phosphatase 2A

Nanobody Mediated Crystallization of an Archeal Mechanosensitive Channel

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