DE loop in monomeric β2m and in interaction within the MHC-I.

<p>(A) Ribbon representation of monomeric β2m (PDB code 2YXF). The DE loop residues are shown in yellow sticks. (B) Stereo view of the DE loop and Phe56 (yellow sticks) when interacting with the heavy chain in the MHC-I (electrostatic surface and green sticks). Trp60 is establishing a H-bond with Asp122 from the heavy chain (PDB code 4L29).</p

Amyloid fibril formation of wt β2m, D59P, W60G and W60V β2m variants monitored by ThT fluorescence (given in arbitrary units).

<p>Amyloid fibril formation of wt β2m, D59P, W60G and W60V β2m variants monitored by ThT fluorescence (given in arbitrary units).</p

ATR/FTIR characterisation of DE loop mutants in the fibrillar state.

<p>A) The absorption spectra of the D59P fibrils were collected before and after incubation in D₂O for different times. Spectra are reported in the regions of Amide I, Amide II, and Amide II’ bands. Arrows point to the spectral changes at increased incubation time in D₂O. Absorption spectra are normalized at the Amide I maximum. B) Second derivatives of the absorption spectra of (A) in the Amide I region. The spectra collected after D₂O additions were normalized at the tyrosine band [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122449#pone.0122449.ref027" target="_blank">27</a>]. The peak positions of the main components are indicated. C) The absorption spectra of the W60V fibrils were collected before and after incubation in D₂O for different times and reported as in (A). D) Second derivatives of the absorption spectra of (C) in the Amide I region. E) Time course of the peak positions of the main intermolecular β-sheet component of wt, D59P, and W60V amyloid fibrils are reported after D₂O addition to the protein films. Error bars represent the standard deviation of at least three independent fibril preparations. The peak positions were taken from the second derivative spectra. F) The absorption spectra of W60G, wt, D59P, and W60V fibrils and that of W60G supernatant are reported in the Amide I region. The intermolecular β-sheet structure absorption band is marked. G) Second derivative spectra of the W60G supernatant collected before and after 23 hours from D₂O addition. The peak positions of the main components are indicated.</p

Reciprocal Influence of Protein Domains in the Cold-Adapted Acyl Aminoacyl Peptidase from <em>Sporosarcina psychrophila</em>

<div><p>Acyl aminoacyl peptidases are two-domain proteins composed by a C-terminal catalytic α/β-hydrolase domain and by an N-terminal β-propeller domain connected through a structural element that is at the N-terminus in sequence but participates in the 3D structure of the C-domain. We investigated about the structural and functional interplay between the two domains and the bridge structure (in this case a single helix named α1-helix) in the cold-adapted enzyme from <em>Sporosarcina psychrophila</em> (SpAAP) using both protein variants in which entire domains were deleted and proteins carrying substitutions in the α1-helix. We found that in this enzyme the inter-domain connection dramatically affects the stability of both the whole enzyme and the β-propeller. The α1-helix is required for the stability of the intact protein, as in other enzymes of the same family; however in this psychrophilic enzyme only, it destabilizes the isolated β-propeller. A single charged residue (E10) in the α1-helix plays a major role for the stability of the whole structure. Overall, a strict interaction of the SpAAP domains seems to be mandatory for the preservation of their reciprocal structural integrity and may witness their co-evolution.</p> </div

Kinetic stability of SpAAP variants.

<p>Freshly purified proteins were incubated at 50°C, before determining residual activities on <i>p</i>NP-But (<b>A</b>) and Nac-leu-<i>p</i>NA (<b>B</b>). Activity at t₀ was taken as 100%. Wild-type SpAAP (diamonds), K6A (squares), E10A (cross plus), R14A (stars), K6A-E10A (triangles), K6A-E10A-R14A (circles). Experiments were carried out in triplicate and error bars presented in the plot.</p

ATR/FTIR characterisation of wt β2m in the native and the fibrillar state.

<p>A) The absorption spectra of the native β2m in form of a protein film were collected before and after incubation in D₂O for different times. Spectra are reported in the regions of Amide I (AI), Amide II (AII), and Amide II’ (AII’). Arrows point at increasing incubation time in D₂O. Absorption spectra are normalized at the Amide I maximum. B) Second derivatives of the absorption spectra of (A) in the Amide I region. The spectra collected after D₂O addition were normalized at the tyrosine band [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122449#pone.0122449.ref027" target="_blank">27</a>]. The marked peak positions of the two components due to the native antiparallel β-sheet structures refer to the spectrum of the undeuterated sample. C) Time course of the peak position of the main native β-sheet component reported after D₂O addition to the protein film. Error bars represent the standard deviation of three independent samples. The peak positions were taken from the second derivative spectra. D) Absorption spectra of the wt β2m fibrils collected before and after incubation in D₂O, reported as in (A). E) Second derivatives of the absorption spectra of (D) in the Amide I region. Spectra of two undeuterated fibrils obtained from independent preparations are compared to show fibril heterogeneity. The spectra collected after D₂O addition were normalized at the tyrosine band [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122449#pone.0122449.ref027" target="_blank">27</a>]. The peak positions of the main components are indicated. F) Time course of the peak position of the main intermolecular β-sheet component is reported after D₂O addition. Error bars represent the standard deviation of three independent fibril preparations. The peak positions were taken from the second derivative spectra.</p

AFM characterisation of wt β2m and DE loop mutants aggregates incubated for one week.

<p>Tapping mode AFM images (height data) of mature fibrils of wt β2m and DE loop mutants obtained after one week incubation. Scan size 1.2 μm; the scale bars correspond to a Z range of: A and D) 55 nm; B) 70 nm; C) 65 nm. E-H) histograms of fibril height measured from fibril cross-sectional profiles in the topographic AFM images.</p

Thermal stability of Nterm domain investigated by FTIR and intrinsic fluorescence analyses.

<p>FTIR second derivative spectra of Nterm-SpAAP (<b>A</b>) and Δα-Nterm-SpAAP (<b>B</b>) collected at different times of incubation at 50°C. Arrows point to increased times of incubation. (<b>C</b>) Time dependence of the intensity variation of the native β-sheet component at ∼ 1636 cm⁻¹, taken from the second derivative peak-intensity of Nterm-SpAAP (filled circles) and Δα-Nterm-SpAAP (empty circles). (<b>D</b>) Thermal denaturation of Nterm-SpAAP (filled circles) and Δα-Nterm-SpAAP (empty circles) was monitored from 20°C to 95°C by intrinsic fluorescence (excitation at 280 nm) and fitted to a two-state model.</p

Design of SpAAP variants and analysis of their solubility.

<p>(<b>A</b>) Domain organization of SpAAP and schematic representation of the deletion mutants analyzed in this study. (<b>B, C</b>) SDS-PAGE analysis of solubility of SpAAP variants produced in <i>E. coli</i> cells. Frozen cell pellets were handled at room temperature to separate the soluble and insoluble protein fractions. For each sample, total cell extracts (ET), soluble proteins (SOL) and insoluble proteins (INS) were extracted from the same amount of cells (0.12 OD₆₀₀). (<b>B</b>) Samples from cells producing the wild-type protein (wt), the protein deleted of the N-terminal helix (Δα), the isolated β-propeller (Nter), the isolated catalytic domain (Cter). (<b>C</b>) Samples from cells producing the charge mutants K6A, E10A, R14A. The solubility profile of all single-charge mutants were similar and are not shown. MW: molecular weight marker.</p

ATR/FTIR characterisation of DE loop mutants in the fibrillar state.

<p>A) The absorption spectra of the D59P fibrils were collected before and after incubation in D₂O for different times. Spectra are reported in the regions of Amide I, Amide II, and Amide II’ bands. Arrows point to the spectral changes at increased incubation time in D₂O. Absorption spectra are normalized at the Amide I maximum. B) Second derivatives of the absorption spectra of (A) in the Amide I region. The spectra collected after D₂O additions were normalized at the tyrosine band [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122449#pone.0122449.ref027" target="_blank">27</a>]. The peak positions of the main components are indicated. C) The absorption spectra of the W60V fibrils were collected before and after incubation in D₂O for different times and reported as in (A). D) Second derivatives of the absorption spectra of (C) in the Amide I region. E) Time course of the peak positions of the main intermolecular β-sheet component of wt, D59P, and W60V amyloid fibrils are reported after D₂O addition to the protein films. Error bars represent the standard deviation of at least three independent fibril preparations. The peak positions were taken from the second derivative spectra. F) The absorption spectra of W60G, wt, D59P, and W60V fibrils and that of W60G supernatant are reported in the Amide I region. The intermolecular β-sheet structure absorption band is marked. G) Second derivative spectra of the W60G supernatant collected before and after 23 hours from D₂O addition. The peak positions of the main components are indicated.</p