Quenched H/D exchange mapped onto the 3D structure of Aβ(1–42) fibrils.

<p>(A) The disease-relevant structure is composed of two molecules per fibril layer [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172862#pone.0172862.ref014" target="_blank">14</a>] (pdb code 2NAO). The double horse shoe-like core structure of the Aβ(1–42) fibrils shows the highest protection. (B) Structure of the polymorph by Lührs et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172862#pone.0172862.ref016" target="_blank">16</a>] (pdb code 2beg). Solvent protected residues are color coded in red, solvent exposed residues in blue, residues with 30–50% protection in orange, and residues lacking data in white.</p

Relaxation Matrix Analysis of Spin Diffusion for the NMR Structure Calculation with eNOEs

NMR structure determination is usually based on distance restraints extracted semiquantitatively from cross peak volumes or intensities in NOESY spectra. The recent introduction of exact NOEs (eNOE) by Vogeli et al. opens an avenue for the ensemble-based structure determination of proteins on the basis of eNOE-derived quantitative distance restraints. We present an approach to extract eNOE from build-up curve intensities. For the determination of eNOEs, spin diffusion is a major source of errors. A full relaxation matrix analysis is used to calculate the spin diffusion contribution to the NOESY cross peaks of each individual spin pair of interest. A software program is written, which requires as input the peak intensities from the various NOESY spectra as well as a 3D structure of the protein. This structure can be either an X-ray structure or an NMR structure determined with the conventional approach. The outputs of the program are the eNOE rates, the autorelaxation rates, as well as graphs and quality factors from the individual NOE build-up curves for semiautomated analysis of the derived rates. The protocol is straightforward, and the program integrates well into the current structure calculation workflow

Structural differences of Aβ(1–42) fibrils of this study (A and B) and of Aβ(1–42)^Mox fibrils of Lührs et al. (C and D) [16].

<p>They are revealed by the amide exchange rates <i>k</i>_ex / h^-1 of individual ¹⁵N-¹H moieties and the relative population <i>P</i>(F) of the slow and fast exchanging components of the exchange. The exchange rates and population P(F) of Aβ(1–42) fibrils of this study (A and B, colored in grey and black) have been extracted from the H/D exchange curves in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172862#pone.0172862.g001" target="_blank">Fig 1</a> and compared with corresponding values of the polymorph studied by Lührs et al. (C and D, colored in grey and black). The blue arrows are indicating the β-strands determined by solid-state NMR of Aβ(1–42) fibrils and the one defined by Lührs et al. and the dashed line indicates missing data. Since the color code follows the main population, following the black colored data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172862#pone.0172862.g001" target="_blank">Fig 1A</a> elucidates immediately, that the main population undergoes fast exchange between residues 1–13, slow exchange between residues 16–22, fast exchange between 23–27 (with the exception of V24), slow exchange between 28–36, fast exchange between 37–39 (with no data for G38), slow exchange between 40–41, and fast exchange for residue 42.</p

Residue-resolved quenched H/D exchange data for the identification of solvent protected ¹⁵N-¹H- moieties of Aβ(1–42) fibrils.

<p>The relative peak intensities of the [¹⁵N,¹H]-HMQC spectra (y-axis) acquired in the DMSO solution back predicted to time zero are shown for each exchange time in D₂O as indicated along the x-axis. Smooth solid lines represent the mono- exponential fits of the data points. Some residues (for example: H6, G9, Y10, and G25) show a clear decay towards zero indicative of fast exchange, whereas others (for example F19, F20, I31, and I32) remain at high intensity indicative of a high solvent protection. For many residues the exchange is composed of a fast and a slow exchanging component indicative of the presence of two distinct populations.</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 [¹⁵N,¹H]-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 [¹⁵N,¹H]-TROSY spectrum opening an avenue for the structural studies of a helical membrane protein in a bilayer system by solution state NMR.</p> </div

YgaP⁻ incorporation into DMPC nanodiscs.

<p>(<b>A</b>) Size exclusion chromatography (Superdex 200 10/300GL) of YgaP⁻ 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⁻ in MSP1/DMPC nanodiscs. (<b>B</b>) SDS-PAGE of YgaP⁻ 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 [¹⁵N,¹H]-TROSY spectra of ²H,¹⁵N-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 [¹⁵N,¹H]-TROSY spectrum of ²H,¹⁵N-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 [¹⁵N,¹H]-TROSY spectrum of ²H,¹⁵N-labeled YgaP.</p

Effects of DHPC-7/LMPG mixed micelles on the N-terminal rhodanese domain and full length YgaP

<p>⁻<b>.</b> (<b>A</b>) 2D [¹⁵N,¹H]-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 [¹⁵N,¹H]-TROSY spectra of ²H,¹⁵N-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⁻ in DHPC-7/LMPG. 4–12% NuPAGE Bis-Tris gel (Invitrogen, Carslbad). Lanes: (MW) SeeBlue plus2 prestained (Invitrogen, Carslbad), (1) YgaP⁻ 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⁻ 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

NMR spectra of YgaP in various micellar systems as indicated.

<p>2D [¹⁵N,¹H]-TROSY spectra of ²H,¹⁵N-labeled YgaP⁻ 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 [¹⁵N,¹H]-TROSY of the N-terminal rhodanese domain of YgaP. The individual cross peaks are labeled according to the sequential assignment. (<b>G</b>) ¹H and ¹⁵N chemical shift differences (labeled Δδ¹H^N and Δδ¹⁵N) between the N-terminal rhodanese domain in solution and the N-terminal rhodanese domain of full length YgaP⁻ 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 ¹H^N and <b>Δ</b>δ¹⁵N chemical shift differences indicates the same tertiary structure of the rhodanese domain in solution and in presence of mixed micelles.</p

CD spectroscopy of Nogo-A-Δ20 at 25°C.

<p>Recombinant Nogo-A-Δ20 exhibits a spectrum typical for unstructured proteins. Addition of FC12, thought to mimic a membrane environment, slightly enhances the structural composition of the protein. However, the observed changes are negligible compared to the α-helical structure reported for Nogo-66 upon FC12 addition [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161813#pone.0161813.ref019" target="_blank">19</a>]. Addition of ECL2 to Nogo-A-Δ20 in a membrane-mimicking environment does not lead to folding, either.</p

Activity assay of ¹³C, ¹⁵N-labelled Nogo-A-Δ20.

<p>3T3 fibroblasts were plated on Nogo-A-Δ20 or control substrate for 1 h and fixed with paraformaldehyde. Non-linear regression reveals an IC₅₀ value of ~40 pmol/cm². Mean cell size ± standard deviation from three wells is shown for each concentration.</p