Comparison of the apparent N-degron strength generated and measured by different methods.

<p>Apparent N-degron strength ordered from high to low destabilizing activity. Pulse chase data were obtained by Bachmaier et al., 1989, fluorescent timer-based measurements by Khmaelinski et al., 2012, fluorescence-based measurements during this study. A color code indicates whether an amino acid is a primary (dark cyan), secondary (blue) or tertiary (red) destabilizing residue at the amino-terminus of a protein, or if it is stabilizing (gold) in the absence of N-acetylation.</p

Structural comparison of the TEV protease with the R203G mutant.

<p><b>A</b>) Ribbon structure of the TEV protease (green) was overlaid with the mutant (dark cyan). The structure of the R203G mutant, which corresponds to R345G in the pTEV⁺ protease, was obtained by homology modeling using an x-ray structure of the TEV protease as template. Views from three different sides are shown. The residues of the catalytic triad H46, D81, and C151 are indicated (TEV protease: blue; mutant: magenta). The two arginine residues close to the catalytic center (R49, R50) are shown in yellow (TEV protease) and light magenta (G203 mutant). The R203 residue is shown in orange, the G203 in red. The two β-sheets, which are mentioned in the text that close the catalytic center are marked by asterisks. <b>B</b>) Surface charge distribution of the TEV protease compared to the R203G mutant. Surface charges were calculated using the software package MolMol. Positive charge is represented by blue color, negative charge by red color.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

<i>In vivo</i> analysis of the P1' Specificity of the pTEV2 protease.

<p><b>A</b>) Processing of the tester constructs CFP-TDegX-RFP (plasmid encoded) was observed after induction of pTEV2 protease production (P_<i>GAL1</i><i>-pTEV2</i> in yeast strain YCR56). Conditions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067915#pone-0067915-g001" target="_blank">Figure 1B</a>. <b>B</b>) Quantification of the P1' Specificity of the pTEV2 protease. Decrease of full length tester construct after two hours was normalized to initial values and relative efficiency normalized to proline was calculated (cleavage efficiency = ([X]_2h/[Pro]_2h×100-100) ×(−1)), assuming that the recognition sequence with proline at the P1’ Position is not cleaved at all. For each construct two immunoblotting experiments were quantified. Values for constructs with Arg and Phe at the P1’ Position cleaved by the pTEV⁺ protease obtained at the same time are shown as reference. Yeast strains YCR56 (pTEV2 protease production) or YCT1169 (pTEV⁺ protease production) harboring plasmid-based constructs were used for the measurements. <b>C</b>) Quantification of X-RFP depletion. The RFP fluorescence was analyzed by fluorimeter measurements after induction of pTEV2 protease synthesis (upper graph, conditions as in Figure 1C) and the depletion efficiency was calculated (error bars: SEM of at least three experiments). Same constructs as in B. The difference between the arginine construct cleaved by pTEV2 and pTEV⁺ protease is very significant (unpaired t test; p = 0.007).</p

Generation of a TEV protease that cleaves efficiently the recognition sequence ENLYFQ-R.

<p><b>A</b>) Scheme of the construct used for the screening procedure: The bidirectional degron module GFP-cODC1-TDegX-RFP (X = F or R) was fused to the phosphoribosylaminoimidazole carboxylase Ade2. Cleavage by the TEV protease leads to activation of the C-degron cODC1 and the N-degron TDegX resulting in proteasomal degradation of Ade2-GFP-cODC1 as well as TDegX-RFP. <b>B</b>) Test for adenine biosynthesis in cells bearing different degron constructs fused chromosomally to <i>ADE2</i>. The yeast strains (ESM356-1, YCT1266, and YCR8) were grown in patches on solid media (YPD, YP+galactose, yeast nitrogen base + 2% glucose, and yeast nitrogen base + 2% galactose; from left to right). <b>C</b>) Scheme illustrating the mutagenesis and selection procedure to obtain a TEV protease which efficiently processes the recognition sequence ENLYFQ-R (left side). The plate is an example to show the difference in color of clones with efficient proteolysis of ENLYFQ-R (red colonies) and clones with insufficient proteolysis (white colonies). Please note that the high degree of red colonies was obtained because the R345G mutant was generated already in the first round of mutagenesis and enriched in subsequent rounds. <b>D</b>) Expression of <i>pTEV2 protease</i> (plasmid-based, R345G mutant) using the <i>GAL1</i> promoter induces the adenine auxotrophy phenotype in <i>ade2-GFP-cODC1-TDegR-RFP</i> cells (YCR6). Serial dilutions (1:10) were grown on solid media as in B.</p

Influence of the P2' residue on substrate degradation.

<p><b>A</b>) Analysis of tester construct proteolysis and depletion as well as TEV protease production by immunoblotting. Tester constructs (plasmid based): CFP-TDegXY-RFP, XY=RH, RL, X corresponds to the P1' position, Y to the P2' position; proteases: pTEV⁺ (yeast strain YCT1169), pTEV2 (YCR56). Conditions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067915#pone-0067915-g001" target="_blank">Figure 1B</a>; antibodies directed against tRFP, GFP, TEV, and Tub1 (loading control) were used to obtain the immunoblot. <b>B</b>) The RFP fluorescence of the tester constructs CFP-TDegXY-RFP was followed over time after induction of TEV protease synthesis by fluorimeter measurements (three measurements for each construct; error bars indicate the standard error of the mean; same constructs as in A).</p

Environmental Conditions Modulate the Switch among Different States of the Hydrophobin Vmh2 from Pleurotus ostreatus

Fungal hydrophobins are amphipathic, highly surface-active, and self-assembling proteins. The class I hydrophobin Vmh2 from the basidiomycete fungus Pleurotus ostreatus seems to be the most hydrophobic hydrophobin characterized so far. Structural and functional properties of the protein as a function of the environmental conditions have been determined. At least three distinct phenomena can occur, being modulated by the environmental conditions: (1) when the pH increases or in the presence of Ca²⁺ ions, an assembled state, β-sheet rich, is formed; (2) when the solvent polarity increases, the protein shows an increased tendency to reach hydrophobic/hydrophilic interfaces, with no detectable conformational change; and (3) when a reversible conformational change and reversible aggregation occur at high temperature. Modulation of the Vmh2 conformational/aggregation features by changing the environmental conditions can be very useful in view of the potential protein applications

SEC chromatograms of K113N BS-RNase aggregates obtained by lyophilising the protein from a 40% (v/v) acetic acid solution.

<p>(<b>A</b>), Superdex 75 chromatogram of the mutant (blue) overlapped with a chromatogram of wt BS-RNase (red). (<b>B</b>), SEC of K113N BS-TT₁ and TT₂ gathered together immediately after their elution from the aggregates mixture (day 0-blue curve) and re-cromatographed after storage in 0.2 M NaPi, pH 6.7, for one, two, or three days (1-red, 2-dark green, 3-pink curves, respectively).</p

SEC chromatograms and PAGE under non denaturing conditions of BS-RNase aggregates obtained by lyophilising the protein from a 40% (v/v) acetic acid solution.

<p>(<b>A</b>) SEC pattern obtained with a Sephadex G100 column. Elution with ammonium acetate 0.1 M, pH 5.65, flow rate of 0.4 ml/min. (<b>B</b>) SEC chromatogram of BS-RNase multimers superimposed with that of RNase A oligomers: both patterns were obtained with a Superdex 75 10/300 GL column. Elution with 0.2 M NaPi, pH 6.7, flow rate 0.1 ml/min. (<b>C</b>) Enlarged Superdex 75 SEC pattern of BS-RNase aggregates; in the inset, 7.5% non denaturing PAGE of the two BS-tetramers, run-time 110 min. (<b>D</b>) Additional purification of the two BS-RNase tetramers: their mixture was concentrated to 25 µl in 0.4 M NaPi, and re-chromatographed in the Superdex 75 column equilibrated with the same buffer (dashed+dotted line). Then, TT₁ and TT₂ fractions were further purified: once for TT₁, continuous line; twice for TT₂, dotted and dashed lines, respectively. In the right part of the panel are reported the models of two N-swapped BS-RNase tetramers proposed by Adinolfi <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046804#pone.0046804-Adinolfi1" target="_blank">[13]</a>: they cannot be associated to both tetramers. The various BS-RNase species are: D, native dimer; TT₁ and TT₂, two tetrameric conformers, H (1 and 2), hexamers; L.O., larger oligomers. Concerning RNase A, grey italics labels: <i>M</i>, native monomer, <i>N_D</i>, N-terminal-swapped dimer, <i>C_D</i>, C-terminal-swapped dimer; <i>T</i>, trimers; <i>NCN_TT</i>: double N+C-swapped tetramer; <i>CNC_TT</i>: double C+N-swapped tetramer; <i>P*</i>: pentamers; <i>H*</i>: hexamers. The asterisk* is present to mention that P and H positions are derived from data obtained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046804#pone.0046804-Gotte1" target="_blank">[21]</a>.</p

Quantification and structural features of BS-RNase oligomers.

a<p>Calculated from DLS analysis.</p>b<p>The elution volumes of the BS-tetramers derive from their additional SEC purification with 0.4 M NaPi as eluent (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046804#pone-0046804-g001" target="_blank">Figure 1D</a>).</p>c<p>L.O.: mixture of BS-RNase octamers and larger oligomers.</p