Supporting evidence for an alternative mouse PAPSS2 N-terminus.

<p>The mouse EST database at GenBank was searched using BLASTN using the following exon2 sequence of murine PAPS synthase: <i>gaccagcaaa aatccaccaa tgtggtctac caggcccatc atgtgagcag gaacaagaga ggacaagtgg ttggaaccag gggaggattc cgaggatgta ccgtgtggct aaca</i>.</p><p>All 15 hits are listed in this table. Moreover, the available cDNAs are listed. EST, expressed sequence tag.</p

A positively charged helical structure in PAPSS1 shows export activity.

<p><b>A</b>, alignment of residues Ser102-Gly124 of PAPSS1 (P1) and the respective Ser92-Gly114 sequence of PAPSS2 (P2). The two arginine residues targeted by alanine mutation are highlighted. NPS secondary structure consensus prediction confirms an extended α-helical conformation; “.”, coil; h/H, weak/prominent helical propensity. <b>B</b>, mapping of this motif (cyan) on the interface of the APS kinase dimer within the PAPSS1 crystal structure 1X6V. The two arginine residues are shown in stick representation (blue). Additionally, Glu108 is shown in stick representation (cyan). This residue may be necessary for charge compensation. <b>C</b>, a recombinant GST-PAPSS1-Helix-GFP protein showed export activity upon microinjection into the nuclei of Vero cells at the indicated time points (left panels). In contrast, mutation of two arginine residues completely abolished nuclear export (middle panel), and the respective substrate remained nuclear as microinjected GST-GFP alone under the same experimental conditions (right panel). As a positive control, the Rev NES of HIV (LQLPPLERLTL) was completely exported from the nucleus within 30 min following microinjection. Approximately 50 cells were injected, and representative examples are shown.</p

Localisation of PAPS synthase wild-type fusion proteins.

<p><b>A and B</b>, microscopic images of PAPSS1- and PAPSS2-EGFP fusions. Transfected HeLa cells were fixed upon 24 h of fluorescent protein expression. Within every sample, cells with varying protein localisation could be observed. <b>C</b>, schematic for classifying localisation of PAPS synthase fusion proteins and their mutants. <b>D</b>, localisation pattern for PAPS synthase wild-type proteins with respect to the way of fixation and the expression time. A total of 1026 cells for PAPSS1-EGFP (P1) and 1942 cells for PAPSS2B-EGFP (P2) were evaluated, respectively, in fixed HeLa cells after 24 hours of expression. For all other conditions at least 200 cells were scored except PAPSS2-EGFP in living HeLa cells (n = 122). EGFP, enhanced green fluorescent protein.</p

Apparent molecular weight of PAPSS1 mutants.

<p>All proteins were loaded at a concentration of 2–10 µM on the gel filtration column. Dilution during chromatography is three-fold at most. Co-localisation was assessed after binarisation of images as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029559#s3" target="_blank">methods</a> section.</p

A conserved NLS within the N-terminus of PAPS synthases.

<p><b>A</b>, alignment of the N-terminal protein sequence of PAPS synthases from several organisms. PAPS synthase N-termini were aligned with regard to their conserved N-terminal KKxK motif and the beginning of the conserved sequence of the globular APS kinase domain. Although this motif should be regarded as K(K/R)x(K/R) only due to the sequences from fruitfly and worm, we adhere to KKxK in the text for simplicity. All positively charged amino acids are written in red and underlined. An exon-exon border is indicated for all sequences derived from Ensembl. For mouse PAPS synthase 2 an alternative N-terminal sequence, P2 mouse*, is given that is supported by 14 different EST sequences. The sequence for the swapping construct of the PAPSS1 N-terminal sequence and the remaining part (body) of PAPSS2 is given below. Sequences used: human PAPSS1 (P1) [RefSeq: NM_005443], mouse P1 [RefSeq: NM_011863], rat P1 [RefSeq: NM_001106471], medaka P1 [Ensembl: ENSORLP00000008587], splice form RA from <i>Drosophila melanogaster</i> [FlyBase: FBgn0020389], the PPS-1 protein from <i>C. elegans</i> [RefSeq: NM_069456/Wormbase: T14G10.1], medaka P2 [Ensembl: ENSORLG00000006251], rat P2 [RefSeq: NM_001106375], mouse P2 [RefSeq: NM_011864<b>.3</b>], mouse P2* (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029559#pone.0029559.s001" target="_blank">Information S1</a></b>) and human PAPSS2B [RefSeq: NM_001015880]. <b>B</b>, HeLa cells expressing PAPS synthase point mutants within the conserved KKxK motif as EGFP fusions. All samples were fixed after 24 h expression. <b>C</b>, quantitative evaluation of mutants shown in B and comparison to the wild-type proteins PAPSS1-EGFP and PAPSS2-EGFP. <b>D and E</b>, a swapping construct with the N-terminal sequence of PAPSS1 and the PAPSS2-EGFP body expressed in HeLa cells shows clear nuclear accumulation relative to the PAPSS2-EGFP wild type.</p

Impact of the Nanoparticle–Protein Corona on Colloidal Stability and Protein Structure

In biological fluids, proteins may associate with nanoparticles (NPs), leading to the formation of a so-called “protein corona” largely defining the biological identity of the particle. Here, we present a novel approach to assess apparent binding affinities for the adsorption/desorption of proteins to silver NPs based on the impact of the corona formation on the agglomeration kinetics of the colloid. Affinities derived from circular dichroism measurements complement these results, simultaneously elucidating structural changes in the adsorbed protein. Employing human serum albumin as a model, apparent affinities in the nanomolar regime resulted from both approaches. Collectively, our findings now allow discrimination between the formation of protein mono- and multilayers on NP surfaces

The KKxK motif shows import activity.

<p>Two peptide variants of the N-terminus of human PAPSS1 differing in length (long and short) as well as the peptides encoded by the first exon of murine PAPSS2 were tested as GST-GFP transport substrates in microinjection experiments. <b>A</b>, peptide sequences of the transport substrates tested. <b>B</b>, both recombinant GST-PAPSS1_N-term-GFP proteins showed import activity upon microinjection into the nuclei of Vero cells at the indicated time points (left panels). In contrast, an N-terminal peptide derived from murine PAPSS2 (mP2) remained cytosolic as microinjected GST-GFP alone under the same experimental conditions, whereas an N-terminal peptide of an alternatively spliced murine PAPSS2 (mP2*) displayed weak import activity (middle panel). As a positive control, the nuclear localisation signal of the SV40 T-antigen (SV40NLS) was completely imported into the nucleus within 60 min following microinjection (right panel). Approximately 50 cells were injected, and representative examples are shown.</p

Effect of LMB treatment on PAPS synthase localisation.

<p><b>A</b>, localisation patterns for PAPS synthase wild-type fusion proteins following LMB treatment. HeLa cells expressing PAPSS1- and PAPSS2-EGFP fusion proteins were treated with the export inhibitor leptomycin B (LMB) at a concentration of 10 nM for prolonged periods of time. At least 200 cells were visually evaluated per lane according to the scheme from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029559#pone-0029559-g001" target="_blank">figure 1</a>. <b>B</b>, examples of PAPSS1-EGFP expressing HeLa cells after 5 h LMB or mock treatment demonstrating increased nuclear accumulation caused by LMB. <b>C</b>, alternative assessment of quantitative localisation after binarisation. Original images are shown before and after binarisation. In this instance, the extracted parameters were: area of Hoechst fluorescence (16%), EGFP fluorescence (8.5%) and co-localisation (3.8%), co-localisation relative to Hoechst (23%) and EGFP (44%) areas (Coloc_relH and Coloc_relE, respectively) as well as the ratio of EGFP and Hoechst areas (0.53) reflecting transformation efficiency. The area of Coloc_relE was found to be the most robust parameter for nuclear localisation of our PAPS synthase-EGFP fusion proteins. <b>D</b>, effect of LMB treatment on PAPS synthase-EGFP fusion protein localisation measured according to <b>C</b>. The area of Coloc_relE significantly increases for both PAPSS1- and PAPSS2-EGFP proteins upon prolonged LMB treatment. *p values for unpaired two-tailed t-test relative to no LMB treatment: PAPSS1-EGFP with 3 h LMB (0.034) and 5 h LMB (0.006) as well as for PAPSS2B-EGFP with 5 h LMB treatment (0.038).</p

Heterogeneity of localisation does not correlate with PAPS synthase-EGFP expression levels.

<p><b>A</b>, microscopic images of fixed HeLa cells transfected with PAPSS1- and PAPSS2-EGFP fusion plasmids were analysed on a single-cell level using ImageJ 1.45. Therefore, cells were scored according to the schematic of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029559#pone-0029559-g001" target="_blank">Figure 1</a> and cell borders were defined as regions of interest (ROI). Then, cell surface as well as mean fluorescence intensity was measured separately for each cell followed by background correction. <b>B</b>, using single cell analysis, 147 cells were measured by for PAPSS1-EGFP and 150 for PAPSS2-EGFP. The classification of these ensembles recapitulates the heterogeneous localisation pattern for both PAPS synthases reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029559#pone-0029559-g001" target="_blank">Figure 1</a>. <b>C</b>, multiplication of cell area and mean fluorescence intensity results in a dimension-less fluorescence. This value represents the total fluorescence of each cell and is assumed to correlate with the expression level of EGFP fusion proteins. For the three major classes of each ensemble, fluorescence was plotted in a box-and-whisker plot. No correlation between expression level/fluorescence and cellular localisation could be derived.</p

Recognition of a Flexible Protein Loop in Taspase 1 by Multivalent Supramolecular Tweezers

Many natural proteins contain flexible loops utilizing well-defined complementary surface regions of their interacting partners and usually undergo major structural rearrangements to allow perfect binding. The molecular recognition of such flexible structures is still highly challenging due to the inherent conformational dynamics. Notably, protein–protein interactions are on the other hand characterized by a multivalent display of complementary binding partners to enhance molecular affinity and specificity. Imitating this natural concept, we here report the rational design of advanced multivalent supramolecular tweezers that allow addressing two lysine and arginine clusters on a flexible protein surface loop. The protease Taspase 1, which is involved in cancer development, carries a basic bipartite nuclear localization signal (NLS) and thus interacts with Importin α, a prerequisite for proteolytic activation. Newly established synthesis routes enabled us to covalently fuse several tweezer molecules into multivalent NLS ligands. The resulting bi- up to pentavalent constructs were then systematically compared in comprehensive biochemical assays. In this series, the stepwise increase in valency was robustly reflected by the ligands’ gradually enhanced potency to disrupt the interaction of Taspase 1 with Importin α, correlated with both higher binding affinity and inhibition of proteolytic activity