Activation of Tyr, AzF, and AmF by TyrRS and the AzRSs.

<p>Tyr, AzF and AmF were used at a concentration of 5 mM. No amino acid was added to the negative control (w/o aa). TyrRS (A) was added at 1 µM and AzRS1 (B), AzRS3 (C), and AzRS6 (D) at a concentration of 5 µM. The data for each o-aaRS were collected in one series of experiments (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031992#s4" target="_blank">Materials and Methods</a> for details). The average of duplicate determinations is shown; the bars indicate the discrete values.</p

Structures of tyrosine and its analogs used in this study.

<p>Structures, names and abbreviations are shown.</p

ESI-MS analyses of selected hSOD1 variants.

<p>Only variant proteins for which defined mass spectra were obtained are shown. The same hSOD1 variants were detected on the immunoblot in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031992#pone-0031992-g002" target="_blank">Figure 2</a>. The corresponding ESI-MS spectra are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031992#pone-0031992-g003" target="_blank">Figure 3</a>. All hSOD1 variants were found with the N-terminal methionine cleaved off and acetylated alanine at position 2, as reported in the literature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031992#pone.0031992-Hallewell1" target="_blank">[41]</a>. The occasionally attached sodium ions (+22.99 Da) most probably originated from the <i>Strep</i>-Tactin elution buffer which contained 150 mM NaCl. The buffer was not exchanged during sample concentration in order to avoid protein loss. In some of the protein preparations we found a known disulfide bond (S-S, −2 Da; between C57 and C146 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031992#pone.0031992-Lindberg1" target="_blank">[70]</a>).</p>1<p>All hSOD1 masses were calculated without N-terminal methionine, acetylated alanine at position 2 and with completely reduced cysteines.</p

Activation of Tyr, Bpa, AzF, and AmF by BpaRS.

<p>Tyr, Bpa, AzF and AmF were used at a concentration of 5 mM. In the negative control, the amino acid was omitted (w/o aa). BpaRS was added at a concentration of 3 µM. The data were all recorded in one row of experiments and each value was determined in duplicate. The bars denote the discrete values.</p

Fluorescence recovery of EGFP and (4<i>S</i>)-FPro-EGFP.

<p>The proteins were denatured by boiling (95°C, 5 min) in 8 M urea and refolded by 100-fold dilution into the buffer without urea (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001680#s3" target="_blank">Methods</a> section for details). Fluorescence emission profiles of (A) (4<i>S</i>)-FPro-EGFP and (B) EGFP upon excitation of the chromophore at 488 nm before denaturation and after 24 h refolding at room temperature. (4<i>S</i>)-FPro-EGFP recovers more than 95% of its fluorescence before denaturation, whereas EGFP recovers only up to 60% of its initial fluorescence (this is in agreement with literature data). (C) The refolding kinetics of both proteins starts with an initial fast phase that is followed by a slow refolding phase. (4<i>S</i>)-FPro-EGFP refolds approximately 2 times faster than EGFP. The percentage of refolding was calculated on the basis of the final constant amount of fluorescence, corresponding to 100% of refolding. Normalized fluorescence in arbitrary units (au) was plotted against time.</p

Fluoroproline variants of EGFP.

<p>(A) Characteristic β-barrel structure of EGFP with the 10 Pro residues highlighted. Cro66 indicates the fluorophore. (B) Chemical structures of proline and the two proline analogs, (2<i>S</i>,4<i>S</i>)-4-fluoroproline ((4<i>S</i>)-FPro), and (2<i>S</i>,4<i>R</i>)-4-fluoroproline ((4<i>R</i>)-FPro). (C) Expression profile of EGFP and its 4-FPro variants in <i>E. coli</i>. EGFP and (4<i>S</i>)-FPro-EGFP are predominantly soluble, whereas (4<i>R</i>)-FPro-EGFP is insoluble. Purified EGFP was applied as the molecular weight marker (M) and is indicated by the arrow; S, soluble protein fraction; I, insoluble protein fraction. Proteins were separated by SDS-PAGE and stained with Coomassie Brillant Blue.</p

X-ray structure of the proline-rich pentapeptide (4<i>S</i>)-FPro54-Val55-(4<i>S</i>)-FPro56-Trp57-(4<i>S</i>)-FPro58 (PVPWP).

<p>The continuous electron density (grey, 2Fo-Fc; contouring levels 1 σ) indicates fluorine atoms at the 4<i>S</i>-position in three buried Pro residues (54, 56, 58). Their experimental electron densities are localized unambiguously (image preparation with PYMOL (<a href="http://pymol.sourceforge.net/" target="_blank">http://pymol.sourceforge.net/</a>)). Out of the three Pro residues forming <i>trans</i> peptide bonds, only Pro56 exhibits predominant C^γ-<i>exo</i> pucker whereas the other two have pyrrolidine rings with C^γ-<i>endo</i> conformation. The rigid local secondary structure of this motif forces the (4<i>S</i>)-fluorinated pyrrolidine ring of (4<i>S</i>)-FPro56 into a stereochemically unfavorable C^γ-<i>exo</i> pucker.</p

Stereo image of the crystal structure of (4<i>S</i>)-FPro-EGFP.

<p>All Pro residues were replaced by (4<i>S</i>)-FPro. Fluoroprolines (13, 54, 56, 58, 75, 89, 187, 192, 196, 211) as well as the C- and N-termini (C, N) are indicated. The chromophore is shown in green and fluorines in cyan. Note that all fluorinated Pro residues except (4<i>S</i>)-FPro56 exhibit a C^γ-<i>endo</i> pucker. Only (4<i>S</i>)-FPro56 shows a C^γ-<i>exo</i> puckered pyrrolidine ring.</p

Clickable Shiga Toxin B Subunit for Drug Delivery in Cancer Therapy

In recent years, receptor-mediated drug delivery has gained major attention in the treatment of cancer. The pathogen-derived Shiga Toxin B subunit (STxB) can be used as a carrier that detects the tumor-associated glycosphingolipid globotriaosylceramide (Gb3) receptors. While drug conjugation via lysine or cysteine offers random drug attachment to carriers, click chemistry has the potential to improve the engineering of delivery systems as the site specificity can eliminate interference with the active binding site of tumor ligands. We present the production of recombinant STxB in its wild-type (STxBwt) version or incorporating the noncanonical amino acid azido lysine (STxBAzK). The STxBwt and STxBAzK were manufactured using a growth-decoupled Escherichia coli (E. coli)-based expression strain and analyzed via flow cytometry for Gb3 receptor recognition and specificity on two human colorectal adenocarcinoma cell linesHT-29 and LS-174characterized by high and low Gb3 abundance, respectively. Furthermore, STxBAzK was clicked to the antineoplastic agent monomethyl auristatin E (MMAE) and evaluated in cell-killing assays for its ability to deliver the drug to Gb3-expressing tumor cells. The STxBAzK–MMAE conjugate induced uptake and release of the MMAE drug in Gb3-positive tumor cells, reaching 94% of HT-29 cell elimination at 72 h post-treatment and low nanomolar doses while sparing LS-174 cells. STxBAzK is therefore presented as a well-functioning drug carrier, with a possible application in cancer therapy. This research demonstrates the feasibility of lectin carriers used in delivering drugs to tumor cells, with prospects for improved cancer therapy in terms of straightforward drug attachment and effective cancer cell elimination

Effect of Noncanonical Amino Acids on Protein–Carbohydrate Interactions: Structure, Dynamics, and Carbohydrate Affinity of a Lectin Engineered with Fluorinated Tryptophan Analogs

Protein–carbohydrate interactions play crucial roles in biology. Understanding and modifying these interactions is of major interest for fighting many diseases. We took a synthetic biology approach and incorporated noncanonical amino acids into a bacterial lectin to modulate its interactions with carbohydrates. We focused on tryptophan, which is prevalent in carbohydrate binding sites. The exchange of the tryptophan residues with analogs fluorinated at different positions resulted in three distinctly fluorinated variants of the lectin from <i>Ralstonia solanacearum</i>. We observed differences in stability and affinity toward fucosylated glycans and rationalized them by X-ray and modeling studies. While fluorination decreased the aromaticity of the indole ring and, therefore, the strength of carbohydrate–aromatic interactions, additional weak hydrogen bonds were formed between fluorine and the ligand hydroxyl groups. Our approach opens new possibilities to engineer carbohydrate receptors