Electron cryomicroscopy analysis of infectious prion protein amyloid fibrils.

<p><b>(A)</b> Section of a cryo electron micrograph showing prion fibrils lacking the glycosylphosphatidylinositol (GPI) anchor. A single isolated and twisted fibril used for the 3-D reconstruction is enclosed by a black box. <b>(B)</b> Close-up view of the isolated prion fibril. <b>(C)</b> Reprojected image of the 3-D fibril map for comparison with the unprocessed image (B). <b>(D)</b> 3-D reconstruction of the GPI-anchorless prion fibril. <b>(E)</b> Cross section of the reconstructed fibril showing two distinct protofilaments. <b>(F)</b> Contoured density maps of the cross section with lines contoured at increasing levels of 0.125 σ. <b>(G)</b> Cartoon depicting the proposed configuration of the polypeptide chains in the prion fibril. Please note that this is not an atomistic model. <b>(H)</b> Close-up view of the possible ß-sheet stacking in a four-rung ß-solenoid architecture for illustration purposes only. Different colors represent different ß-solenoid rungs. Characteristic distances of the four-rung ß-solenoid architecture are labeled. Figure adapted from [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006229#ppat.1006229.ref010" target="_blank">10</a>].</p

Infectious prions and prion-like proteins.

<p>Infectious prions and prion-like proteins.</p

Internal dynamics.

<p><b>A. </b>¹⁵N linewidths of perdeuterated hHP1β as a function of residue number, measured from a TROSY-HSQC recorded at 700 MHz and 303 K. <b>B.</b> R_ex values of CD in full-length hHP1β as a function of residue number. <b>C.</b> Comparison of steady-state ¹H-¹⁵N heteronuclear NOE values of CD in full-length hHP1β in the free state (black circles) with those of CD in full-length hHP1β in complex with the H3K_C9me3 peptide (1–15) at a molar ratio of 1∶4 (white circles). Both measurements were performed at 298 K, 600 MHz proton Larmor frequency, 5 s recycle delay, on a 0.3 mM ¹⁵N-perdeuterated hHP1β sample.</p

Rotational diffusion tensor of CD and CSD within full-length hHP1β.

<p>Diffusion tensor parameters for the different tumbling models, obtained from experimental ¹⁵N spin-relaxation data through ROTDIF, are listed: the overall rotational correlation time τ_c; D_xx, D_yy and D_zz are the principal values of the diffusion tensor; Q is the quality factor defined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060887#pone.0060887-Walker1" target="_blank">[27]</a>; P defines the probability that an improvement in the fit when a more complex model is applied has occurred by chance. The best model for CD and CSD is marked in bold. The little improvement in the fit with the more complex fully-anisotropic model was not statistically significant for CD.</p

Structural Plasticity in Human Heterochromatin Protein 1β

<div><p>As essential components of the molecular machine assembling heterochromatin in eukaryotes, HP1 (Heterochromatin Protein 1) proteins are key regulators of genome function. While several high-resolution structures of the two globular regions of HP1, chromo and chromoshadow domains, in their free form or in complex with recognition-motif peptides are available, less is known about the conformational behavior of the full-length protein. Here, we used NMR spectroscopy in combination with small angle X-ray scattering and dynamic light scattering to characterize the dynamic and structural properties of full-length human HP1β (hHP1β) in solution. We show that the hinge region is highly flexible and enables a largely unrestricted spatial search by the two globular domains for their binding partners. In addition, the binding pockets within the chromo and chromoshadow domains experience internal dynamics that can be useful for the versatile recognition of different binding partners. In particular, we provide evidence for the presence of a distinct structural propensity in free hHP1β that prepares a binding-competent interface for the formation of the intermolecular β-sheet with methylated histone H3. The structural plasticity of hHP1β supports its ability to bind and connect a wide variety of binding partners in epigenetic processes.</p> </div

Weakening of hydrogen bonds in supercooled solution.

<p>Ratios of ^h3J_NC’ trans-hydrogen bond scalar couplings at 278 K and 298 K (A), and at 270 K and 278 K (B). Only residues not affected by signal overlap were included. Errors were calculated on the basis of the signal-to-noise ratio of the cross and reference peak. On the x-axis the donor and acceptor residue are indicated.</p

¹H-¹⁵N chemical shift changes of specific residues in comparison to the chemically denatured state.

<p>Selected regions of 2D [¹H,¹⁵N]-HSQCs with decreasing temperature: 298 K (red), 288 K (blue), 278 K (green), 268 K (maroon), and 260 K (purple). The unfolded state of ubiquitin was obtained by addition of 8 M urea at pH 2 (shown in thick grey).</p

hHP1β populates an extended ensemble.

<p><b>A.</b> Dynamic light scattering of hHP1β. The histogram plot shows the experimental data from one measurement consisting of 20 acquisitions. The diffusion coefficient value (D) reported is an average of five measurements done at identical conditions. <b>B.</b> PFG-NMR based diffusion plot of hHP1β. The natural logarithm of the intensity ratio I/I₀ linearly correlates (R = 0.98) with Q, a combined parameter dependent on the gradients strength and delays as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060887#pone.0060887-Zheng1" target="_blank">[51]</a>. For the intensity ratio, four integrated signals in the 2.4–0.7 ppm region were measured from 32 spectra recorded with increasing gradient strength from 5–75% of the maximum value. Diffusion coefficient values (D) from NMR and DLS experiments were converted into hydrodynamic radius (R_h) values based on the Stokes-Einstein’s equation. <b>C.</b> Small angle X-ray scattering profile of hHP1β. The plot displays the decimal logarithm of the scattering intensity as a function of momentum transfer, s. The distance distribution function is displayed in the inset. <b>D. </b><i>R_g</i> distributions from EOM for hHP1β: initial random pool (continuous line) and selected ensembles averaged over 50 independent EOM runs (dashed line).</p

Non-linearity of the [¹H,¹⁵N] chemical shift changes in ubiquitin at decreasing temperatures.

<p>(A) 2D [¹H,¹⁵N]-HSQCs of ubiquitin for temperatures from 298 K to 263 K. Selected resonance assignments are indicated. (B–E) Weighted average [¹H,¹⁵N] chemical shift changes as a function of temperature for selected residues. The red line shows the straight line fit to the data in the range 298K-273K. Differences between amide proton (F) and nitrogen (G) temperature coefficients in the range 273 K-263 K and 298 K-273 K. The location of helices and β-strands is schematically shown above.</p

Ubiquitin remains folded in supercooled solution down to 263 K.

<p>(A, B) Correlation between experimental backbone [¹H,¹⁵N] residual dipolar couplings observed at (A) 270 K and (B) at 278 K with values calculated by singular-value decomposition from the solution NMR structure of ubiquitin (PDB entry 1D3Z). Residues deviating from a linear fit are marked.</p