The ribosome stabilizes partially folded intermediates of a nascent multi-domain protein.

Co-translational folding is crucial to ensure the production of biologically active proteins. The ribosome can alter the folding pathways of nascent polypeptide chains, yet a structural understanding remains largely inaccessible experimentally. We have developed site-specific labelling of nascent chains to detect and measure, using 19F nuclear magnetic resonance (NMR) spectroscopy, multiple states accessed by an immunoglobulin-like domain within a tandem repeat protein during biosynthesis. By examining ribosomes arrested at different stages during translation of this common structural motif, we observe highly broadened NMR resonances attributable to two previously unidentified intermediates, which are stably populated across a wide folding transition. Using molecular dynamics simulations and corroborated by cryo-electron microscopy, we obtain models of these partially folded states, enabling experimental verification of a ribosome-binding site that contributes to their high stabilities. We thus demonstrate a mechanism by which the ribosome could thermodynamically regulate folding and other co-translational processes.

Discovery and Characterization of a New Cold-Active Protease From an Extremophilic Bacterium via Comparative Genome Analysis and in vitro Expression.

Following a screening of Antarctic glacier forefield-bacteria for novel cold-active enzymes, a psychrophilic strain Psychrobacter sp. 94-6PB was selected for further characterization of enzymatic activities. The strain produced lipases and proteases in the temperature range of 4-18°C. The coding sequence of an extracellular serine-protease was then identified via comparative analysis across Psychrobacter sp. genomes, PCR-amplified in our strain 94-6PB and expressed in the heterologous host E. coli. The purified enzyme (80 kDa) resulted to be a cold-active alkaline protease, performing best at temperatures of 20-30°C and pH 7-9. It was stable in presence of common inhibitors [ß-mercaptoethanol (ß-ME), dithiothreitol (DTT), urea, phenylmethylsulfonyl fluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA)] and compatible with detergents and surfactants (Tween 20, Tween 80, hydrogen peroxide and Triton X-100). Because of these properties, the P94-6PB protease may be suitable for use in a new generation of laundry products for cold washing. Furthermore, we assessed the microdiversity of this enzyme in Psychrobacter organisms from different cold habitats and found several gene clusters that correlated with specific ecological niches. We then discussed the role of habitat specialization in shaping the biodiversity of proteins and enzymes and anticipate far-reaching implications for the search of novel variants of biotechnological products.

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence.

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore's conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced "cyan" variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.