20 research outputs found
YgaP<sup>−</sup> incorporation into DMPC nanodiscs.
<p>(<b>A</b>) Size exclusion chromatography (Superdex 200 10/300GL) of YgaP<sup>−</sup> in 20 mM bis-Tris-HCl pH 7, 150 mM NaCl, 3 mM DHPC-7, 1 mM LMPG, and 5 mM TCEP. (upper panel) and YgaP<sup>−</sup> in MSP1/DMPC nanodiscs. (<b>B</b>) SDS-PAGE of YgaP<sup>−</sup> in DMPC nanodiscs. 12% NuPAGE Bis-Tris gel (Invitrogen, Carslbad). Lanes: (MW) SeeBlue plus2 prestained (Invitrogen, Carslbad), (1)-(3) Different dilutions of the YgaP/DMPC nanodisc reaction mixture in the SDS sample buffer after the removal of detergents by Biobeads to resolve the partial overlap due to apparent over-staining in lane 1 for the individual identification of YgaP and MSP1 as indicated. (<b>C</b>)–(<b>D</b>) 2D [<sup>15</sup>N,<sup>1</sup>H]-TROSY spectra of <sup>2</sup>H,<sup>15</sup>N-labeled YgaP purified in 6 mM DHPC-7 and 1 mM LMPG and (<b>C</b>) incorporated in DMPC nanodiscs or (<b>D</b>) in nanodiscs with deuterated d-54 DMPC. (<b>E</b>) 2D [<sup>15</sup>N,<sup>1</sup>H]-TROSY spectrum of <sup>2</sup>H,<sup>15</sup>N-labeled YgaP purified in 3 mM FC12. The sample of (E) was used for a DMPC nanodisc preparation as shown in (<b>F</b>): 2D [<sup>15</sup>N,<sup>1</sup>H]-TROSY spectrum of <sup>2</sup>H,<sup>15</sup>N-labeled YgaP.</p
Detergent/Nanodisc Screening for High-Resolution NMR Studies of an Integral Membrane Protein Containing a Cytoplasmic Domain
<div><p>Because membrane proteins need to be extracted from their natural environment and reconstituted in artificial milieus for the 3D structure determination by X-ray crystallography or NMR, the search for membrane mimetic that conserve the native structure and functional activities remains challenging. We demonstrate here a detergent/nanodisc screening study by NMR of the bacterial α-helical membrane protein YgaP containing a cytoplasmic rhodanese domain. The analysis of 2D [<sup>15</sup>N,<sup>1</sup>H]-TROSY spectra shows that only a careful usage of low amounts of mixed detergents did not perturb the cytoplasmic domain while solubilizing in parallel the transmembrane segments with good spectral quality. In contrast, the incorporation of YgaP into nanodiscs appeared to be straightforward and yielded a surprisingly high quality [<sup>15</sup>N,<sup>1</sup>H]-TROSY spectrum opening an avenue for the structural studies of a helical membrane protein in a bilayer system by solution state NMR.</p> </div
NMR spectra of YgaP in various micellar systems as indicated.
<p>2D [<sup>15</sup>N,<sup>1</sup>H]-TROSY spectra of <sup>2</sup>H,<sup>15</sup>N-labeled YgaP<sup>−</sup> in (<b>A</b>) FC12, (<b>B</b>) DHPC-7, (<b>C</b>) DHPC-7 and FC12, (<b>D, F</b>) DHPC-7 and LMPG. (<b>E</b>) 2D [<sup>15</sup>N,<sup>1</sup>H]-TROSY of the N-terminal rhodanese domain of YgaP. The individual cross peaks are labeled according to the sequential assignment. (<b>G</b>) <sup>1</sup>H and <sup>15</sup>N chemical shift differences (labeled Δδ<sup>1</sup>H<sup>N</sup> and Δδ<sup>15</sup>N) between the N-terminal rhodanese domain in solution and the N-terminal rhodanese domain of full length YgaP<sup>−</sup> in the optimized mixed micellar conditions (i.e. 6 mM DHPC, 1 mM LMPG). The lack of profound up- or down-filed <b>Δ</b>δ <sup>1</sup>H<sup>N</sup> and <b>Δ</b>δ<sup>15</sup>N chemical shift differences indicates the same tertiary structure of the rhodanese domain in solution and in presence of mixed micelles.</p
Effects of DHPC-7/LMPG mixed micelles on the N-terminal rhodanese domain and full length YgaP
<p><sup>−</sup><b>.</b> (<b>A</b>) 2D [<sup>15</sup>N,<sup>1</sup>H]-TROSY spectra of the N-terminal rhodanese domain in absence (Black) and in presence of 9 mM DHPC-7, 2 mM LMPG (Red). For better clarity a portion of the spectrum is magnified as indicated. (<b>B</b>) 2D [<sup>15</sup>N,<sup>1</sup>H]-TROSY spectra of <sup>2</sup>H,<sup>15</sup>N-labeled of YgaP with optimum detergent concentration (i.e. 6 mM DHPC, 1 mM LMPG) (Black) and in presence of 9 mM DHPC-7, and 2 mM LMPG, respectively (Red). Black arrows indicate regions of the red spectrum where resonances are missing, indicating the effect of detergent excess in the quality of the spectrum. (<b>C</b>) SDS-PAGE of the nickel affinity purification of YgaP<sup>−</sup> in DHPC-7/LMPG. 4–12% NuPAGE Bis-Tris gel (Invitrogen, Carslbad). Lanes: (MW) SeeBlue plus2 prestained (Invitrogen, Carslbad), (1) YgaP<sup>−</sup> after membrane extraction in DHPC-7/LMPG micelles, (2) Loading flow-through fraction of Nickel resin, (3) Washing of Nickel resin, (4) Elution of YgaP<sup>−</sup> with buffer containing 500 mM imidazole (details of the buffer used are given in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054378#s2" target="_blank">Material and Methods</a> section).</p
Intermolecular Detergent–Membrane Protein NOEs for the Characterization of the Dynamics of Membrane Protein–Detergent Complexes
The interaction between membrane
proteins and lipids or lipid mimetics
such as detergents is key for the three-dimensional structure and
dynamics of membrane proteins. In NMR-based structural studies of
membrane proteins, qualitative analysis of intermolecular nuclear
Overhauser enhancements (NOEs) or paramagnetic resonance enhancement
are used in general to identify the transmembrane segments of a membrane
protein. Here, we employed a quantitative characterization of intermolecular
NOEs between <sup>1</sup>H of the detergent and <sup>1</sup>H<sup>N</sup> of <sup>2</sup>H-perdeuterated, <sup>15</sup>N-labeled α-helical
membrane protein–detergent complexes following the exact NOE
(eNOE) approach. Structural considerations suggest that these intermolecular
NOEs should show a helical-wheel-type behavior along a transmembrane
helix or a membrane-attached helix within a membrane protein as experimentally
demonstrated for the complete influenza hemagglutinin fusion domain
HAfp23. The partial absence of such a NOE pattern along the amino
acid sequence as shown for a truncated variant of HAfp23 and for the <i>Escherichia coli</i> inner membrane protein YidH indicates the
presence of large tertiary structure fluctuations such as an opening
between helices or the presence of large rotational dynamics of the
helices. Detergent–protein NOEs thus appear to be a straightforward
probe for a qualitative characterization of structural and dynamical
properties of membrane proteins embedded in detergent micelles
Expression and Functional Characterization of Membrane-Integrated Mammalian Corticotropin Releasing Factor Receptors 1 and 2 in <i>Escherichia coli</i>
<div><p>Corticotropin-Releasing Factor Receptors (CRFRs) are class B1 G-protein-coupled receptors, which bind peptides of the corticotropin releasing factor family and are key mediators in the stress response. In order to dissect the receptors' binding specificity and enable structural studies, full-length human CRFR1α and mouse CRFR2β as well as fragments lacking the N-terminal extracellular domain, were overproduced in <i>E. coli</i>. The characteristics of different CRFR2β -PhoA gene fusion products expressed in bacteria were found to be in agreement with the predicted ones in the hepta-helical membrane topology model. Recombinant histidine-tagged CRFR1α and CRFR2β expression levels and bacterial subcellular localization were evaluated by cell fractionation and Western blot analysis. Protein expression parameters were assessed, including the influence of <i>E. coli</i> bacterial hosts, culture media and the impact of either PelB or DsbA signal peptide. In general, the large majority of receptor proteins became inserted in the bacterial membrane. Across all experimental conditions significantly more CRFR2β product was obtained in comparison to CRFR1α. Following a detergent screen analysis, bacterial membranes containing CRFR1α and CRFR2β were best solubilized with the zwitterionic detergent FC-14. Binding of different peptide ligands to CRFR1α and CRFR2β membrane fractions were similar, in part, to the complex pharmacology observed in eukaryotic cells. We suggest that our <i>E. coli</i> expression system producing functional CRFRs will be useful for large-scale expression of these receptors for structural studies.</p></div
Influence of signal peptides on the expression of CRFRs.
<p>Comparative analysis of hCRFR1α (A) and mCRFR2β (B) constructs encoding no signal peptide (No SP), PelB signal peptide or DsbA signal peptide. Expressions were carried out in TB medium with Rosetta2(DE3) strain and the samples were analyzed by Western blot with His<sub>6</sub>-tag antibody. For each expression vector tested, 1 µl of IB and M fractions were loaded on the gel; the dilution factor of each sample is indicated.</p
Detergent screen for solubilization of CRFRs.
<p>The efficacy of 12 different detergents, or detergent mixes, in solubilizing PelB-hCRFR1α (<b>A</b>) and PelB-mCRFR2β (<b>B</b>) from bacterial membranes was evaluated, after overnight incubation, by phase separation via ultracentrifugation. The resultant soluble fractions were subjected to His<sub>6</sub>-tag antibody Western blot analysis. As a control, an equivalent aliquot of the original membrane fraction (M), which had not been subjected to solubilization, was loaded on the gel. The irregular spot present in the lower part of the B panel is due to a non-specific contamination. For abbreviations of detergent molecules see main text.</p
Alkaline phosphatase fusion protein analysis in <i>E. coli</i> of mCRFR2β.
<p>(<b>A</b>) Specifically designed C-terminally truncated versions of mCRFR2β fused to bacterial membrane topology reporter alkaline phosphatase (PhoA) confer different phenotypes at the level of colony color. PhoA activity was assessed qualitatively by visual inspection of the colonies. (<b>B</b>) The bacterial colony colors conferred by these different protein fusions are in agreement with the hepta-helical transmembrane model of mCRFR2β. The aa numeration refers to the native receptors pre-protein sequence.</p
Influence of various <i>E. coli</i> host strain on the expression of CRFRs.
<p>Expressions of PelB-hCRFR1α (<b>A</b>) and PelB-mCRFR2β (<b>B</b>) were carried out in LB medium in four different strains. Equivalent volumes of the bacterial inclusion bodies and membrane fractions were analyzed by Western blot with His<sub>6</sub>-tag antibody. Parental strain BL21(DE3) transformed with the same constructs but treated without IPTG inducer was used as a negative control.</p