Modulating bilayer mechanical properties to promote the coupled folding and insertion of an integral membrane protein

Herrmann, Michaela; Danielczak, Bartholomäus; Textor, Martin; Klement, Jessica; Keller, Sandro

doi:10.1007/s00249-015-1032-y

Cited by 16 publications

(14 citation statements)

References 53 publications

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“…Figure 2a shows the elution profile of monomeric OmpLA refolded into LDAO [ 35 ] as monitored by UV absorbance, LS, and RI detectors. Data were recorded according to the procedure described in the protocol below.…”

Section: Resultsmentioning

confidence: 99%

Characterising protein/detergent complexes by triple-detection size-exclusion chromatography

2016

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BackgroundIn vitro investigations of membrane proteins usually depend on detergents for protein solubilisation and stabilisation. The amount of detergent bound to a membrane protein is relevant to successful experiment design and data analysis but is often unknown. Triple-detection size-exclusion chromatography enables simultaneous separation of protein/detergent complexes and protein-free detergent micelles and determination of their molar masses in a straightforward and absolute manner. Size-exclusion chromatography is used to separate different species, while ultraviolet absorbance, static light scattering, and refractive index measurements allow molar mass determination of protein and detergent components.ResultsWe refined standard experimental and data-analysis procedures for challenging membrane-protein samples that elude routine approaches. The general procedures including preparatory steps, measurements, and data analysis for the characterisation of both routine and complex samples in difficult solvents such as concentrated denaturant solutions are demonstrated. The applicability of the protocol but also its limitations and possible solutions are discussed, and an extensive troubleshooting section is provided.ConclusionsWe established and validated a protocol for triple-detection size-exclusion chromatography that enables the inexperienced user to perform and analyse measurements of well-behaved protein/detergent complexes. More experienced users are provided with an example of a more sophisticated analysis procedure allowing mass determination under challenging separation conditions.

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Section: Resultsmentioning

confidence: 99%

Characterising protein/detergent complexes by triple-detection size-exclusion chromatography

2016

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“…For POPC/chol membrane this could be related with the fact that cholesterol increases the order of the POPC fluid membrane rendering it less prone to receptor insertion. The increase in the reconstitution rate taking place in POPC/SM/Chol membrane might be due to the fact that membranes with lateral heterogeneity present places of low surface tension or defects (in the contacts between the different domains) that can be opportunistically used by the receptor to insert in a facilitated manner46. Alternatively it is also possible that the receptors inserts faster in brain SM rich domains that have increased thickness compared to disordered domains that might better match the receptor hydrophobic portion.…”

Section: Discussionmentioning

confidence: 99%

Real time monitoring of membrane GPCR reconstitution by plasmon waveguide resonance: on the role of lipids

Calmet

Maria

Harté

et al. 2016

Sci Rep

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G-protein coupled receptors (GPCRs) are important therapeutic targets since more than 40% of the drugs on the market exert their action through these proteins. To decipher the molecular mechanisms of activation and signaling, GPCRs often need to be isolated and reconstituted from a detergent-solubilized state into a well-defined and controllable lipid model system. Several methods exist to reconstitute membrane proteins in lipid systems but usually the reconstitution success is tested at the end of the experiment and often by an additional and indirect method. Irrespective of the method used, the reconstitution process is often an intractable and time-consuming trial-and-error procedure. Herein, we present a method that allows directly monitoring the reconstitution of GPCRs in model planar lipid membranes. Plasmon waveguide resonance (PWR) allows following GPCR lipid reconstitution process without any labeling and with high sensitivity. Additionally, the method is ideal to probe the lipid effect on receptor ligand binding as demonstrated by antagonist binding to the chemokine CCR5 receptor.

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“…OmpLA was prepared and purified as described elsewhere (30,31). Briefly, tag-free OmpLA without signal sequence was produced as inclusion bodies (IBs) in Escherichia coli BL21(DE3) cells.…”

Section: Protein Preparationmentioning

confidence: 99%

Slow Interconversion in a Heterogeneous Unfolded-State Ensemble of Outer-Membrane Phospholipase A

Krainer

Gracia

Frotscher

et al. 2017

Biophysical Journal

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Structural and dynamic investigations of unfolded proteins are important for understanding protein-folding mechanisms as well as the interactions of unfolded polypeptide chains with other cell components. In the case of outer-membrane proteins (OMPs), unfolded-state properties are of particular physiological relevance, because these proteins remain unfolded for extended periods of time during their biogenesis and rely on interactions with binding partners to support proper folding. Using a combination of ensemble and single-molecule spectroscopy, we have scrutinized the unfolded state of outer-membrane phospholipase A (OmpLA) to provide a detailed view of its structural dynamics on timescales from nanoseconds to milliseconds. We find that even under strongly denaturing conditions and in the absence of residual secondary structure, OmpLA populates an ensemble of slowly (>100 ms) interconverting and conformationally heterogeneous unfolded states that lack the fast chain-reconfiguration motions expected for an unstructured, fully unfolded chain. The drastically slowed sampling of potentially folding-competent states, as compared with a random-coil polypeptide, may contribute to the slow in vitro folding kinetics observed for many OMPs. In vivo, however, slow intramolecular long-range dynamics might be advantageous for entropically favored binding of unfolded OMPs to chaperones and, by facilitating conformational selection after release from chaperones, for preserving binding-competent conformations before insertion into the outer membrane.

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Modulating bilayer mechanical properties to promote the coupled folding and insertion of an integral membrane protein

Cited by 16 publications

References 53 publications

Characterising protein/detergent complexes by triple-detection size-exclusion chromatography

Characterising protein/detergent complexes by triple-detection size-exclusion chromatography

Real time monitoring of membrane GPCR reconstitution by plasmon waveguide resonance: on the role of lipids

Slow Interconversion in a Heterogeneous Unfolded-State Ensemble of Outer-Membrane Phospholipase A

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