Ultrasensitive Scaffold-Dependent Protease Sensors with Large Dynamic Range

The rational construction of synthetic protein switches with predefined input–output parameters constitutes a key goal of synthetic biology with many potential applications ranging from metabolic engineering to diagnostics. Yet, generally applicable strategies to construct tailor-engineered protein switches have so far remained elusive. Here, we use SpyTag/SpyCatcher-mediated protein ligation to engineer modularly organized, scaffold-dependent protease sensors that exploit a combination of affinity targeting and protease-inducible protein–protein interactions. We use this architecture to create a suite of integrated signal sensing and amplification circuits that can detect the activity of α-thrombin and prostate specific antigen with a dynamic range covering 5 orders of magnitude. We determine the key design features critical for signal transmission between protease-based sensors, transducers, and actuators

Semisynthetic tRNA Complement Mediates <i>in Vitro</i> Protein Synthesis

Genetic code expansion is a key objective of synthetic biology and protein engineering. Most efforts in this direction are focused on reassigning termination or decoding quadruplet codons. While the redundancy of genetic code provides a large number of potentially reassignable codons, their utility is diminished by the inevitable interaction with cognate aminoacyl-tRNAs. To address this problem, we sought to establish an <i>in vitro</i> protein synthesis system with a simplified synthetic tRNA complement, thereby orthogonalizing some of the sense codons. This quantitative i<i>n vitro</i> peptide synthesis assay allowed us to analyze the ability of synthetic tRNAs to decode all of 61 sense codons. We observed that, with the exception of isoacceptors for Asn, Glu, and Ile, the majority of 48 synthetic Escherichia coli tRNAs could support protein translation in the cell-free system. We purified to homogeneity functional Asn, Glu, and Ile tRNAs from the native E. coli tRNA mixture, and by combining them with synthetic tRNAs, we formulated a semisynthetic tRNA complement for all 20 amino acids. We further demonstrated that this tRNA complement could restore the protein translation activity of tRNA-depleted E. coli lysate to a level comparable to that of total native tRNA. To confirm that the developed system could efficiently synthesize long polypeptides, we expressed three different sequences coding for superfolder GFP. This novel semisynthetic translation system is a powerful tool for tRNA engineering and potentially enables the reassignment of at least 9 sense codons coding for Ser, Arg, Leu, Pro, Thr, and Gly

Design and reporting principle of NANOMS.

<p>FRET-biosensor design of the three different NANOMS. (<b>A</b>) The myristoylated N-terminal membrane-targeting motifs of mouse Gα_i2 (residues 1–35), human Yes (1–17)- and human Src (1–16)-kinases were genetically fused to the N-terminus of fluorescent proteins mCFP or mCit. The sequence of the employed membrane-targeting motifs can be found in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066425#pone.0066425.s006" target="_blank">Table S2</a></b>. (<b>B</b>) Intracellular processing involves cleavage of the N-terminal methionine (grey) by methionine amino-peptidase (Met-AP), NMT-mediated myristoylation on glycine 2 (yellow) and depending on the motif cysteine-palmitoylation (red). (<b>C</b>) Lipid modified reporters spontaneously organize into plasma membrane nanocluster. Tight packing of membrane targeted donor (mCFP)- and acceptor (mCit)-fluorophores (blue and yellow squares, respectively) in nanocluster leads to FRET. FRET can decrease due to loss of nanoclustering or cytoplasmic redistribution of the NANOMS after inhibitor treatment. As membrane anchorage is required for the functioning of myristoylated proteins, NANOMS report on functional membrane anchorage.</p

Cherry-picked chemical compound library screen with Yes-NANOMS.

<p>(<b>A</b>) Chemical structures of chemical compounds that were included in the cherry-picked chemical library. (<b>B</b>) BHK21 cells were transfected with Yes-NANOMS and screened with shown chemical compounds at a final concentration of 10 µM/mL. FRET-response of Yes-NANOMS to the chemical compounds is represented with E_max values. Block line indicates the average E_max and the error bars denote the s.e.m (n≥4). Samples were statistically compared with the untreated control. See Methods for more on statistical analysis.</p

The principle of the Ras Recruitment System (RRS).

<p>The system is based on a temperature sensitive GDP exchange factor (encoded by the <i>cdc25–2</i> allele) that is rendered inactive at 36°C trapping endogenous Ras1p in its inactive GDP bound form. Growth is rescued by genetic complementation with a constitutively active mutant of mammalian H-Ras (<i>RAS61</i>). To exert its function and rescue growth, Ras61p needs to be directed to the plasma membrane. This can either occur through protein-protein interactions or lipid modifications such as myristoylation or prenylation. Specifically, prenylation can either be mediated by endogenous protein prenyltransferases (wt-PPTases) that recognise naturally occurring, prenylatable CaaX-box motives or engineered protein prenyltransferases (o-PPTases) that recognise orthogonal CaaX-box motives that are not recognised by the endogenous machinery. For optimal membrane recruitment and genetic complementation in the RRS, the three most C-terminal amino acids of prenylated CaaX-box motives are removed by highly specific protein prenyl proteases located in the endoplasmic reticulum followed by carboxymethylesterification of the C-terminus.</p

Engineering FTases with altered substrate specificities.

<p>(A) CaaX-box motives with positively charged residues in the anchoring position X cannot rescue growth in the RRS and thus provide poor substrates for endogenous FTases in <i>Saccharomyces cerevisiae</i>. (B) Structural model of the αβ-FTase heterodimer derived from <i>Rattus norvegicus</i> (PDB: 1KZO). The C-terminus of the α-subunit (highlighted in blue) is separated by 40 Å from the N-terminus of the β-subunit (highlighted in red). (C) Western blot analysis of GFP-αβ-FTase fusion proteins derived from <i>R</i>. <i>norvegicus</i> expressed in <i>Leishmania tarantolae</i> cell-free expression system. The linker connecting α- and β-subunits contained a TEV protease cleavage site that is cleaved with exogenously added TEV protease. L1: Protein Ladder; L2: Uncleaved GFP-αβ-FTase; L3: GFP-αβ-FTase cleaved with TEV Protease. (D) Fluorescent scan of SDS–PAGE loaded with mCherry-K-Ras <i>in vitro</i> prenylation reaction containing single-chain GFP-αβ-FTase fusion proteins and fluorescent phosphoisoprenoid NBD-GPP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120716#pone.0120716.ref035" target="_blank">35</a>]. Addition of FPP to the reaction prevents formation of the fluorescent reaction product due to competition with the fluorescent lipid donor. L1: Protein Ladder; L2: GFP-αβ-FTase bound to GFP-Cap beads, 5 μM mCherry-K-Ras, 5 μM NBP-GPP; L3: GFP-αβ-FTase bound to GFP-Cap beads, 5 μM mCherry-K-Ras, 5 μM NBD-GPP, 25 μM FPP. (E) To facilitate expression and prevent cross-heterodimerisation between yeast and exogenous FTase subunits, a single-chain αβ-FTase was created based on mutant β-W102T while introducing negative charges at the bottom of the active site at β-G142D and β-G142E enabling FTase to farnesylate a CaaX-box motif with a positive charge in X and thus rescue growth in the RRS. Controls: pYES2 denotes vector control and β-W102T the unmodified, single-chain αβ-FTase^β-W102T mutant neither of which can prenylate the orthogonal CaaX-box motif.</p

NANOMS reports on RNAi-mediated depletion of NMT.

<p>(<b>A</b>) HEK293 EBNA cells transiently expressing Yes-NANOMS and (<b>B</b>) HEK293 cells transiently expressing Gi2-NANOMS were treated with NMT1 or NMT2 specific siRNAs or control siRNA. Knock-down efficiencies are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066425#pone.0066425.s004" target="_blank">Figure S4</a></b>. The characteristic E_max-value was determined on flow cytometric FRET data. The error bars denote the s.e.m (n = 4). Samples were statistically compared with the untreated control. See Methods for more on statistical analysis.</p

NANOMS report on chemical inhibition of NMT.

<p>(<b>A</b>) FRET-responses of Yes-, Src- and Gi2-NANOMS transfected BHK cells treated with 4 µM of the specific NMT inhibitor DDD85646. The error bars denote the s.e.m (n = 5). Samples were statistically compared with the untreated control. See Methods for more information on statistical analysis. (<b>B</b>) Confocal sensitized acceptor FRET-imaging of Yes-NANOMS expressed in BHK cells. Cells were treated as indicated. Top row shows acceptor channel images, and bottom row FRET images. The look-up table shows the FRET-index FR, color coded with high FRET levels in black and yellow (value 1) indicating no FRET. Scale bar is representative for all images and corresponds to 10 µm. (<b>C</b>) Dose-response curves of the effect of DDD85646 on the E_max values of Yes- and Src-NANOMS expressed in BHK cells (n = 6).</p

Summary of constructs employed in this study.

<p>Summary of constructs employed in this study.</p

Mapping CaaX-box dependent membrane recruitment space in yeast.

<p>(A) Flow chart of the mapping experiment. The CaaX-box library was transformed into the RRS screening strain, grown for 4 days under permissive conditions at 25°C, replica plated and then grown for another 4–6 days under restrictive and permissive conditions at 37°C and 25°C. CaaX-box coding plasmid DNA was then isolated and analysed for the two different library sets by NGS with the Ion Torrent system. The enrichment was determined for each of the 8000 different CaaX-box motives by measuring the frequency of each peptide under restrictive conditions and normalising it over its frequency under permissive conditions. (B) Graphic representation of the enrichment factors of 8000 different CaaX-box motives is summarised in a 4D plot: Each axis represents the 20 different amino acids while the size of each dot is proportional to the enrichment of a specific CaaX-box motif. Only CaaX-box motives that have been enriched >3 are shown. (C) Cross-sectional views along the a₂-a₁, X-a₁ and X-a₂ axis illustrate that a₂ exerts the greatest specificity on substrate specificity with small hydrophobic residues highly preferred followed by the anchoring position X and a₁.</p