engineering-golden-fluorescence-selective-pressure-incorporation-non

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 ...

antimicrobial-peptides-produced-selective-pressure-incorporation-non

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all ...

Technical University of Berlin

Technische Universität Berlin

Fluorine being not substantially present in the chemistry of living beings is an attractive element in tailoring novel chemical, biophysical, and pharmacokinetic properties of peptides and proteins. The hallmark of ribosome-mediated artificial amino acid incorporation into peptides and proteins is a broad substrate tolerance, which is assumed to rely on the absence of evolutionary pressure for efficient editing of artificial amino acids. We used the well-characterized editing proficient isoleucyl-tRNA synthetase (IleRS) from  to investigate the crosstalk of aminoacylation and editing activities against fluorinated amino acids. We show that translation of trifluoroethylglycine (TfeGly) into proteins is prevented by hydrolysis of TfeGly-tRNA in the IleRS post-transfer editing domain. The remarkable observation is that dissociation of TfeGly-tRNA from IleRS is significantly slowed down. This finding is in sharp contrast to natural editing reactions by tRNA synthetases wherein fast editing rates for the noncognate substrates are essential to outcompete fast aa-tRNA dissociation rates. Using a post-transfer editing deficient mutant of IleRS (IleRSAla10), we were able to achieve ribosomal incorporation of TfeGly in vivo. Our work expands the knowledge of ribosome-mediated artificial amino acid translation with detailed analysis of natural editing function against an artificial amino acid providing an impulse for further systematic investigations and engineering of the translation and editing of unusual amino acids.

Discovery and Investigation of Natural Editing Function against Artificial Amino Acids in Protein Translation.

The incorporation of non-canonical amino acids (ncAA) is an elegant way for the chemical diversification of recombinantly produced antimicrobial peptides (AMPs). Residue- and site-specific installation methods in several bacterial production hosts hold great promise for the generation of new-to-nature AMPs, and can contribute to tackle the ongoing emergence of antibiotic resistance in pathogens. Especially from a pharmacological point of view, desirable improvements span pH and protease resistance, solubility, oral availability and circulation half-life. Although the primary focus of this report is on ribosomally synthesized and post-translationally modified peptides (RiPPs), we have included selected cases of peptides produced by solid phase peptide synthesis to comparatively show the potential and impact of ncAA introduction. Generally speaking, the introduction of ncAAs in recombinant AMPs delivers novel levels of chemical diversification. Cotranslationally incorporated, they can take part in AMP biogenesis either through direction interaction with elements of the post-translational modification (PTM) machinery or as untargeted sites with unique physicochemical properties and chemical handles for further modification. Together with genetic libraries, genome mining and processing by PTM machineries, ncAAs present not a mere addition to this process, but a highly diverse pool of building blocks to significantly broaden the chemical space of this valuable class of molecules. This perspective summarizes new developments of ncAA containing peptides. Challenges to be resolved in order to reach large-scale pharmaceutical production of these promising compounds and prospects for future developments are discussed.

Prospects of  Incorporation of Non-canonical Amino Acids for the Chemical Diversification of Antimicrobial Peptides.

Marine mussels exhibit potent underwater adhesion abilities under hostile conditions by employing 3,4-dihydroxyphenylalanine (DOPA)-rich mussel adhesive proteins (MAPs). However, their recombinant production is a major biotechnological challenge. Herein, a novel strategy based on genetic code expansion has been developed by engineering efficient aminoacyl-transfer RNA synthetases (aaRSs) for the photocaged noncanonical amino acid ortho-nitrobenzyl DOPA (ONB-DOPA). The engineered ONB-DOPARS enables in vivo production of MAP type 5 site-specifically equipped with multiple instances of ONB-DOPA to yield photocaged, spatiotemporally controlled underwater adhesives. Upon exposure to UV light, these proteins feature elevated wet adhesion properties. This concept offers new perspectives for the production of recombinant bioadhesives.

Photoactivatable Mussel-Based Underwater Adhesive Proteins by an Expanded Genetic Code.

The incorporation of non-canonical amino acids (ncAAs) into ribosomally synthesized and post-translationally modified peptides, e.g., nisin from the Gram-positive bacterium , bears great potential to expand the chemical space of various antimicrobials. The ncAA -Boc-L-lysine (BocK) was chosen for incorporation into nisin using the archaeal pyrrolysyl-tRNA synthetase-tRNA pair to establish orthogonal translation in  for read-through of in-frame amber stop codons. In parallel, recombinant nisin production and orthogonal translation were combined in  cells. Both organisms synthesized bioactive nisin(BocK) variants. Screening of a nisin amber codon library revealed suitable sites for ncAA incorporation and two variants displayed high antimicrobial activity. Orthogonal translation in  and  presents a promising tool to create new-to-nature nisin derivatives.

Tobias Baumann

Institute of Chemistry L 1,
Department of Biocatalysis,
Institute of Chemistry L 1, Department of Biocatalysis

Technical University of Berlin

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

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

Tobias Baumann

Institute of Chemistry L 1,Department of Biocatalysis,Institute of Chemistry L 1, Department of Biocatalysis

Technical University of Berlin

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

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

Institute of Chemistry L 1,
Department of Biocatalysis,
Institute of Chemistry L 1, Department of Biocatalysis