12 research outputs found
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
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
Selection of J147.
<p>CNB-001 (A) is a broadly neuroprotective and neurotrophic derivative of curcumin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027865#pone.0027865-Liu1" target="_blank">[8]</a>. CNB-023 (B) is a derivative of CNB-001 lacking hydroxyl groups but with similar activity that was identified by SAR analysis of CNB-001. The boxed area shows the hypothesized biologically active fragment of CNB-023. A large collection of molecules around this chemical space was generated by the reaction of m-anisaldehyde with 2.4 dimethylphenyl hydrazine (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027865#s4" target="_blank">Materials and Methods</a>) and J147 (C) was selected from the reaction products on the basis of its activity in trophic factor withdrawal (TFW), oxidative stress (oxytosis) and Aβ toxicity assays (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027865#pone-0027865-g002" target="_blank">Figure 2</a>). (D) Final HPLC run of reaction mixture that generated J147. The structure of the most active product (fraction 10) was determined by NMR. J147 was then synthesized, and its crystal structure (insert) determined, confirming its biological activity and structural identity. The following are de-risking and target identification screens, with no significant reproducible hits. J147 was used at 10 micromolar unless indicated. (1) LeadProfiling+P450 screen. Over 60 CNS receptors and transporters (work done by MDS Pharma). (2) hERG (work done by MDS and Absorption Systems). (3) Acute toxicity in rats. Negative at 2 grams/kilogram (work done by Absorption systems). (4) CeeTox “Safe” up to 90 micromolar plasma concentration (work done by CEETOX, INC). (5) 352 protein kinases (done by Ambit). (6) MDRI-MDCK brain penetration classification “High” (work done by Absorption Systems). (7) Enzyme Assays. Cox 1; Cox 2; Lox 5,12,15; Sirt 1,2,3; LT4 hydrolase; LTC4 synthase; cathepsin B; matrix metalloprotease 1; phosphodiesterases 10A1, 11A1, 1A, 2A, 3A, 4A1A, 4B1, 5A, 6, 7A, 7B, 8A1, 9A2; proteasome (done by MDS). (8) Enzyme assays. PPARα, γ; deubiquitinases, BAP1, UCH-L1, L3, USP5; acetylcholine esterase; MAOA; MAOB; phosphatases (19 of them), proteases and caspases (8) (done by Caliper).</p
Preparation of J147.
<p>J147 was prepared as described in the chemistry section of the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027865#s4" target="_blank">materials and methods</a>. The reagents and conditions for each step are as follows: (a) EtOH, rt, 1 h, 90%; (b) (CF<sub>3</sub>CO)<sub>2</sub>O, Et<sub>3</sub>N, 30 min, 10°C, 77%.</p
J147 Increases the Levels of BDNF and BDNF Responsive Protein and Protein Phosphorylation.
<p>(A) BDNF expression in the hippocampus of normal adult rats following two weeks treatment of J147 in food at 200 ppm. J147 treatment increased BDNF levels in the normal rat. Two-tailed t test. *<i>P</i><0.05. (B) BDNF expression in hippocampus of huAPP/PS1 transgenic mice fed J147 for 7 months before sacrifice at 10 months. BDNF levels are decreased in AD transgenic mice and treatment with J147 restores levels to beyond control levels. (C) Homer-1 expression in huAPP/PS1 mice. J147 treatment increases Homer-1 levels. (D) Phosphorylation of PSD95 at Tyr 236, 240. J147 treatment increases phosphorylation of PSD95 in AD transgenic mice. Data are presented as ratio of phospho PSD95 to total PSD95. *<i>P</i><0.05, **<i>P</i><0.01, and ***<i>P</i><0.001. All data shown are means ± SEM. n = 5–6 per group. C = control; C+ = control + J147; A = AD transgenic; A+ = AD transgenic fed J147.</p
Structure activity relationship of a few CNB-001 derivatives.
<p>The structure of CNB-001 is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027865#pone-0027865-g001" target="_blank">Figure 1A</a>. A series of CNB-001 derivatives were synthesized with the goal of removing the hydroxyl group and maintaining and/or improving biological activity in the glutamate based oxytosis assay and extracellular amyloid toxicity. Et (ethyl), Me (Methyl).</p
J147 Reduces Heat Shock Protein Stress Response and Increases Markers for Synaptic Function.
<p>Cell lysates from hippocampal tissue of line 85 mice fed J147 for 7 months were analyzed by Western blotting. C = control; A = AD transgenic; A+ = AD transgenic fed J147. (A) HSP90 levels are decreased in AD transgenic mice fed J147. (B) HOP levels are decreased in AD transgenic mice fed J147. (C) HSP70 levels are increased in AD transgenic mice and these levels are decreased with treatment of J147. (D) Drebrin levels are decreased in AD transgenic mice and J147 treatment restores levels to above control levels. (E) Synapsin-1 levels are decreased in AD transgenic mice and are restored significantly beyond control by J147. (F) Synaptophysin levels are decreased in AD transgenic mice and levels are restored beyond control levels with J147. *<i>P</i><0.05, **<i>P</i><0.01), and ***<i>P</i><0.001. All data shown are means ± SEM, n = 5 per group.</p
J147 Facilitates the Induction of LTP in Schaffer Collateral CA1 Pyramidal Cell Synapses in Rat Hippocampal Slices.
<p>(A) Effect of J147 (1 µM) on basal synaptic transmission. Hippocampal slices were exposed to J147 during the time indicated by the black bar. The fEPSP slope is expressed as the percentage of the value immediately before the addition of J147. J147 does not affect basal synaptic transmission. (B) J147 facilitates the induction of LTP after a weak tetanic stimulation (15 pulses at 100 Hz) which alone does not induce LTP in control slices. The effect of J147 is concentration-dependent. Time course of changes in the fEPSP slope. The hippocampal slices were untreated (o, n = 9) or exposed to J147 (▾, 0.01 µM, n = 6; ▴, 0.1 µM, n = 5; •, 1 µM, n = 7) for the time indicated by the black bar and weak tetanic stimulation was applied at time 0. The fEPSP slope is expressed as the percentage of the value immediately before the application of weak tetanic stimulation. (C) Concentration-dependency. To compare the data among the groups, the averages of the fEPSP slopes 30–60 min after tetanic stimulation were calculated as an index of LTP magnitude. J147 demonstrated a concentration dependent effect with 1000 nM having the greatest effect on the fEPSP slope (n = 7 slices per rat: one-way ANOVA F(3,23) = 4.4, **<i>P</i> = 0.01). (D) Negative Control. −187–88, the alkene form of J147 in which nitrogens are replaced by carbons (o, n = 9; ▴, 0.1 µM, n = 5; •, 1 µM, n = 5) showed no effect. All data shown are means ± SEM.</p