Publications by authors named "Nediljko Budisa"

128 Publications

Biochemistry of fluoroprolines: the prospect of making fluorine a bioelement.

Beilstein J Org Chem 2021 15;17:439-460. Epub 2021 Feb 15.

Department of Chemistry, University of Manitoba, 144 Dysart Rd., Winnipeg, R3T 2N2, Canada.

Due to the heterocyclic structure and distinct conformational profile, proline is unique in the repertoire of the 20 amino acids coded into proteins. Here, we summarize the biochemical work on the replacement of proline with (4)- and (4)-fluoroproline as well as 4,4-difluoroproline in proteins done mainly in the last two decades. We first recapitulate the complex position and biochemical fate of proline in the biochemistry of a cell, discuss the physicochemical properties of fluoroprolines, and overview the attempts to use these amino acids as proline replacements in studies of protein production and folding. Fluorinated proline replacements are able to elevate the protein expression speed and yields and improve the thermodynamic and kinetic folding profiles of individual proteins. In this context, fluoroprolines can be viewed as useful tools in the biotechnological toolbox. As a prospect, we envision that proteome-wide proline-to-fluoroproline substitutions could be possible. We suggest a hypothetical scenario for the use of laboratory evolutionary methods with fluoroprolines as a suitable vehicle to introduce fluorine into living cells. This approach may enable creation of synthetic cells endowed with artificial biodiversity, containing fluorine as a bioelement.
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http://dx.doi.org/10.3762/bjoc.17.40DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934785PMC
February 2021

Multiomics Analysis Provides Insight into the Laboratory Evolution of toward the Metabolic Usage of Fluorinated Indoles.

ACS Cent Sci 2021 Jan 20;7(1):81-92. Epub 2020 Nov 20.

Institute of Chemistry and Biochemistry-Organic Chemistry, Freie Universität Berlin, Berlin 14195, Germany.

Organofluorine compounds are known to be toxic to a broad variety of living beings in different habitats, and chemical fluorination has been historically exploited by mankind for the development of therapeutic drugs or agricultural pesticides. On the other hand, several studies so far have demonstrated that, under appropriate conditions, living systems (in particular bacteria) can tolerate the presence of fluorinated molecules (e.g., amino acids analogues) within their metabolism and even repurpose them as alternative building blocks for the synthesis of cellular macromolecules such as proteins. Understanding the molecular mechanism behind these phenomena would greatly advance approaches to the biotechnological synthesis of recombinant proteins and peptide drugs. However, information about the metabolic effects of long-term exposure of living cells to fluorinated amino acids remains scarce. Hereby, we report the long-term propagation of () in an artificially fluorinated habitat that yielded two strains naturally adapted to live on fluorinated amino acids. In particular, we applied selective pressure to force a tryptophan (Trp)-auxotrophic strain to use either 4- or 5-fluoroindole as essential precursors for the synthesis of Trp analogues, followed by their incorporation in the cellular proteome. We found that full adaptation to both fluorinated Trp analogues requires a low number of genetic mutations but is accompanied by large rearrangements in regulatory networks, membrane integrity, and quality control of protein folding. These findings highlight the cellular mechanisms behind the adaptation to unnatural amino acids and provide the molecular foundation for bioengineering of novel microbial strains for synthetic biology and biotechnology.
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http://dx.doi.org/10.1021/acscentsci.0c00679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7844855PMC
January 2021

Conjugation of Synthetic Polyproline Moietes to Lipid II Binding Fragments of Nisin Yields Active and Stable Antimicrobials.

Front Microbiol 2020 20;11:575334. Epub 2020 Nov 20.

Department of Molecular Genetics, University of Groningen, Groningen, Netherlands.

Coupling functional moieties to lantibiotics offers exciting opportunities to produce novel derivatives with desirable properties enabling new functions and applications. Here, five different synthetic hydrophobic polyproline peptides were conjugated to either nisin AB (the first two rings of nisin) or nisin ABC (the first three rings of nisin) by using click chemistry. The antimicrobial activity of nisin ABC + O6K3 against decreased 8-fold compared to full-length nisin, but its activity was 16-fold better than nisin ABC, suggesting that modifying nisin ABC is a promising strategy to generate semi-synthetic nisin hybrids. In addition, the resulting nisin hybrids are not prone to degradation at the C-terminus, which has been observed for nisin as it can be degraded by nisinase or other proteolytic enzymes. This methodology allows for getting more insight into the possibility of creating semi-synthetic nisin hybrids that maintain antimicrobial activity, in particular when synthetic and non-proteinaceous moieties are used. The success of this approach in creating viable nisin hybrids encourages further exploring the use of different modules, e.g., glycans, lipids, active peptide moieties, and other antimicrobial moieties.
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http://dx.doi.org/10.3389/fmicb.2020.575334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7715017PMC
November 2020

Combating Antimicrobial Resistance With New-To-Nature Lanthipeptides Created by Genetic Code Expansion.

Front Microbiol 2020 5;11:590522. Epub 2020 Nov 5.

Department of Chemistry, University of Manitoba, Winnipeg, MB, Canada.

Due to the rapid emergence of multi-resistant bacterial strains in recent decades, the commercially available effective antibiotics are becoming increasingly limited. On the other hand, widespread antimicrobial peptides (AMPs) such as the lantibiotic nisin has been used worldwide for more than 40 years without the appearance of significant bacterial resistance. Lantibiotics are ribosomally synthesized antimicrobials generated by posttranslational modifications. Their biotechnological production is of particular interest to redesign natural scaffolds improving their pharmaceutical properties, which has great potential for therapeutic use in human medicine and other areas. However, conventional protein engineering methods are limited to 20 canonical amino acids prescribed by the genetic code. Therefore, the expansion of the genetic code as the most advanced approach in Synthetic Biology allows the addition of new amino acid building blocks (non-canonical amino acids, ncAAs) during protein translation. We now have solid proof-of-principle evidence that bioexpression with these novel building blocks enabled lantibiotics with chemical properties transcending those produced by natural evolution. The unique scaffolds with novel structural and functional properties are the result of this bioengineering. Here we will critically examine and evaluate the use of the expanded genetic code and its alternatives in lantibiotics research over the last 7 years. We anticipate that Synthetic Biology, using engineered lantibiotics and even more complex scaffolds will be a promising tool to address an urgent problem of antibiotic resistance, especially in a class of multi-drug resistant microbes known as superbugs.
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http://dx.doi.org/10.3389/fmicb.2020.590522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7674664PMC
November 2020

Courses Based on iGEM/BIOMOD Competitions Are the Ideal Format for Research-Based Learning of Xenobiology.

Chembiochem 2021 Mar 16;22(5):818-825. Epub 2020 Nov 16.

Department of Chemistry, Technische Universität Berlin, Straße des 17. Juni 135, 10623, Berlin, Germany.

Synthetic biology and especially xenobiology, as emerging new fields of science, have reached an intellectual and experimental maturity that makes them suitable for integration into the university curricula of chemical and biological disciplines. Novel scientific fields that include laboratory work are perfect playgrounds for developing highly motivating research-based teaching modules. We believe that research-based learning enriched by digital tools is the best approach for teaching new emerging essentials of academic education. This is especially true when the scientific field as such is still not canonized with text books and best-practice examples. Our experience shows that iGEM/BIOMOD competitions represent an excellent basis for designing research-based courses in xenobiology. Therefore, we present a report on "iGEM-Synthetic Biology" offered at the Technische Universität Berlin as an example.
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http://dx.doi.org/10.1002/cbic.202000614DOI Listing
March 2021

Fine-Tuning Protein Self-Organization by Orthogonal Chemo-Optogenetic Tools.

Angew Chem Int Ed Engl 2021 02 7;60(9):4501-4506. Epub 2021 Jan 7.

Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152, Martinsried, Germany.

A universal gain-of-function approach for the spatiotemporal control of protein activity is highly desirable when reconstituting biological modules in vitro. Here we used orthogonal translation with a photocaged amino acid to map and elucidate molecular mechanisms in the self-organization of the prokaryotic filamentous cell-division protein (FtsZ) that is highly relevant for the assembly of the division ring in bacteria. We masked a tyrosine residue of FtsZ by site-specific incorporation of a photocaged tyrosine analogue. While the mutant still shows self-assembly into filaments, dynamic self-organization into ring patterns can no longer be observed. UV-mediated uncaging revealed that tyrosine 222 is essential for the regulation of the protein's GTPase activity, self-organization, and treadmilling dynamics. Thus, the light-mediated assembly of functional protein modules appears to be a promising minimal-regulation strategy for building up molecular complexity towards a minimal cell.
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http://dx.doi.org/10.1002/anie.202008691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7986231PMC
February 2021

Expanding the Scope of Orthogonal Translation with Pyrrolysyl-tRNA Synthetases Dedicated to Aromatic Amino Acids.

Molecules 2020 Sep 25;25(19). Epub 2020 Sep 25.

Institut für Chemie, Technische Universität Berlin, Müller-Breslau-Straße 10, 10623 Berlin, Germany.

In protein engineering and synthetic biology, pyrrolysyl-tRNA synthetase (PylRS), with its cognate tRNA, is one of the most popular tools for site-specific incorporation of non-canonical amino acids (ncAAs). Numerous orthogonal pairs based on engineered PylRS variants have been developed during the last decade, enabling a substantial genetic code expansion, mainly with aliphatic pyrrolysine analogs. However, comparatively less progress has been made to expand the substrate range of PylRS towards aromatic amino acid residues. Therefore, we set to further expand the substrate scope of orthogonal translation by a semi-rational approach; redesigning the PylRS efficiency. Based on the randomization of residues from the binding pocket and tRNA binding domain, we identify three positions (V401, W417 and S193) crucial for ncAA specificity and enzyme activity. Their systematic mutagenesis enabled us to generate PylRS variants dedicated to tryptophan (such as β-(1-Azulenyl)-l-alanine or 1-methyl-l-tryptophan) and tyrosine (mainly halogenated) analogs. Moreover, our strategy also significantly improves the orthogonal translation efficiency with the previously activated analog 3-benzothienyl-l-alanine. Our study revealed the engineering of both first shell and distant residues to modify substrate specificity as an important strategy to further expand our ability to discover and recruit new ncAAs for orthogonal translation.
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http://dx.doi.org/10.3390/molecules25194418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582959PMC
September 2020

An Engineered Escherichia coli Strain with Synthetic Metabolism for in-Cell Production of Translationally Active Methionine Derivatives.

Chembiochem 2020 Dec 13;21(24):3525-3538. Epub 2020 Oct 13.

Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Sapienza Università di Roma, Piazzale Aldo Moro, 5 - Edificio CU20, 00185, Roma, Italy.

In the last decades, it has become clear that the canonical amino acid repertoire codified by the universal genetic code is not up to the needs of emerging biotechnologies. For this reason, extensive genetic code re-engineering is essential to expand the scope of ribosomal protein translation, leading to reprogrammed microbial cells equipped with an alternative biochemical alphabet to be exploited as potential factories for biotechnological purposes. The prerequisite for this to happen is a continuous intracellular supply of noncanonical amino acids through synthetic metabolism from simple and cheap precursors. We have engineered an Escherichia coli bacterial system that fulfills these requirements through reconfiguration of the methionine biosynthetic pathway and the introduction of an exogenous direct trans-sulfuration pathway. Our metabolic scheme operates in vivo, rescuing intermediates from core cell metabolism and combining them with small bio-orthogonal compounds. Our reprogrammed E. coli strain is capable of the in-cell production of l-azidohomoalanine, which is directly incorporated into proteins in response to methionine codons. We thereby constructed a prototype suitable for economic, versatile, green sustainable chemistry, pushing towards enzyme chemistry and biotechnology-based production.
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http://dx.doi.org/10.1002/cbic.202000257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7756864PMC
December 2020

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

Front Microbiol 2020 13;11:881. Epub 2020 May 13.

GFZ German Research Centre for Geosciences, Helmholtz Centre Potsdam, Section Geomicrobiology, Potsdam, Germany.

Following a screening of Antarctic glacier forefield-bacteria for novel cold-active enzymes, a psychrophilic strain 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 sp. genomes, PCR-amplified in our strain 94-6PB and expressed in the heterologous host . 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 [β-mercaptoethanol (β-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 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.
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http://dx.doi.org/10.3389/fmicb.2020.00881DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7247812PMC
May 2020

Xenobiology: A Journey towards Parallel Life Forms.

Chembiochem 2020 08 22;21(16):2228-2231. Epub 2020 Apr 22.

Biofaction KG, Kundmanngasse 39/12, 1030, Vienna, Austria.

Xenobiology is the science of estranged life forms. More specifically, this is an emergent technoscience that combines advances in genetic engineering with the design of biological systems based on unusual biochemistries delivered by chemical compounds of mostly anthropogenic origin. Xenobiology enables us to create and study strange new life forms, "aliens", not in the way science fiction books do it, but in terms of enlightened science, design, and engineering.
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http://dx.doi.org/10.1002/cbic.202000141DOI Listing
August 2020

Phage capsid nanoparticles with defined ligand arrangement block influenza virus entry.

Nat Nanotechnol 2020 05 30;15(5):373-379. Epub 2020 Mar 30.

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany.

Multivalent interactions at biological interfaces occur frequently in nature and mediate recognition and interactions in essential physiological processes such as cell-to-cell adhesion. Multivalency is also a key principle that allows tight binding between pathogens and host cells during the initial stages of infection. One promising approach to prevent infection is the design of synthetic or semisynthetic multivalent binders that interfere with pathogen adhesion. Here, we present a multivalent binder that is based on a spatially defined arrangement of ligands for the viral spike protein haemagglutinin of the influenza A virus. Complementary experimental and theoretical approaches demonstrate that bacteriophage capsids, which carry host cell haemagglutinin ligands in an arrangement matching the geometry of binding sites of the spike protein, can bind to viruses in a defined multivalent mode. These capsids cover the entire virus envelope, thus preventing its binding to the host cell as visualized by cryo-electron tomography. As a consequence, virus infection can be inhibited in vitro, ex vivo and in vivo. Such highly functionalized capsids present an alternative to strategies that target virus entry by spike-inhibiting antibodies and peptides or that address late steps of the viral replication cycle.
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http://dx.doi.org/10.1038/s41565-020-0660-2DOI Listing
May 2020

The Alanine World Model for the Development of the Amino Acid Repertoire in Protein Biosynthesis.

Int J Mol Sci 2019 Nov 5;20(21). Epub 2019 Nov 5.

Department of Chemistry, University of Manitoba, Dysart Rd. 144, Winnipeg, MB R3T 2N2, Canada.

A central question in the evolution of the modern translation machinery is the origin and chemical ethology of the amino acids prescribed by the genetic code. The RNA World hypothesis postulates that templated protein synthesis has emerged in the transition from RNA to the Protein World. The sequence of these events and principles behind the acquisition of amino acids to this process remain elusive. Here we describe a model for this process by following the scheme previously proposed by Hartman and Smith, which suggests gradual expansion of the coding space as GC-GCA-GCAU genetic code. We point out a correlation of this scheme with the hierarchy of the protein folding. The model follows the sequence of steps in the process of the amino acid recruitment and fits well with the co-evolution and coenzyme handle theories. While the starting set (GC-phase) was responsible for the nucleotide biosynthesis processes, in the second phase alanine-based amino acids (GCA-phase) were recruited from the core metabolism, thereby providing a standard secondary structure, the α-helix. In the final phase (GCAU-phase), the amino acids were appended to the already existing architecture, enabling tertiary fold and membrane interactions. The whole scheme indicates strongly that the choice for the alanine core was done at the GCA-phase, while glycine and proline remained rudiments from the GC-phase. We suggest that the Protein World should rather be considered the Alanine World, as it predominantly relies on the alanine as the core chemical scaffold.
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http://dx.doi.org/10.3390/ijms20215507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6862034PMC
November 2019

Bilayer thickness determines the alignment of model polyproline helices in lipid membranes.

Phys Chem Chem Phys 2019 Oct;21(40):22396-22408

Institute of Chemistry, Technical University of Berlin, Müller-Breslau-Str. 10, Berlin 10623, Germany and Department of Chemistry, University of Manitoba, Dysart Rd. 144, Winnipeg MB R3T 2N2, Canada.

Our understanding of protein folds relies fundamentally on the set of secondary structures found in the proteomes. Yet, there also exist intriguing structures and motifs that are underrepresented in natural biopolymeric systems. One example is the polyproline II helix, which is usually considered to have a polar character and therefore does not form membrane spanning sections of membrane proteins. In our work, we have introduced specially designed polyproline II helices into the hydrophobic membrane milieu and used 19F NMR to monitor the helix alignment in oriented lipid bilayers. Our results show that these artificial hydrophobic peptides can adopt several different alignment states. If the helix is shorter than the thickness of the hydrophobic core of the membrane, it is submerged into the bilayer with its long axis parallel to the membrane plane. The polyproline helix adopts a transmembrane alignment when its length exceeds the bilayer thickness. If the peptide length roughly matches the lipid thickness, a coexistence of both states is observed. We thus show that the lipid thickness plays a determining role in the occurrence of a transmembrane polyproline II helix. We also found that the adaptation of polyproline II helices to hydrophobic mismatch is in some notable aspects different from α-helices. Finally, our results prove that the polyproline II helix is a competent structure for the construction of transmembrane peptide segments, despite the fact that no such motif has ever been reported in natural systems.
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http://dx.doi.org/10.1039/c9cp02996fDOI Listing
October 2019

Anticipating alien cells with alternative genetic codes: away from the alanine world!

Curr Opin Biotechnol 2019 12 3;60:242-249. Epub 2019 Jul 3.

Institute of Chemistry, Technical University of Berlin, Müller-Breslau-Str. 10, Berlin 10623, Germany; Department of Chemistry, University of Manitoba, Dysart Rd .144, Winnipeg R3T 2N2, Manitoba, Canada. Electronic address:

Can we make life with a different genetic amino acid repertoire? Can we expect organisms which would keep newly given genetic code associations permanently? To address these questions, we would like to analyze the existent genetic code amino acid repertoire as formed from derivatives of alanine. Derivation from alanine leads to the α-helix based biological world, the Alanine World, whereas variations in the side-chains enable tertiary folding and subsequent chemical versatility of the proteome. Proline, glycine and pyrrolysine are the rudiments in the current genetic code, indicating that the original set could be different. Furthermore, from the perspective of peptide chemistry, it shall be possible to recruit these alternative scaffolds for the construction of synthetic or alternative life. This would allow for a completely new biological world, potentially as functional and versatile as the existing one. Pursuing these options offers a strategy for a complete re-design or even de-novo creation of living organisms based on entirely different chemical make-up, with completely new set of solutions for both near and distant future biotechnologies.
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http://dx.doi.org/10.1016/j.copbio.2019.05.006DOI Listing
December 2019

Computational Aminoacyl-tRNA Synthetase Library Design for Photocaged Tyrosine.

Int J Mol Sci 2019 May 11;20(9). Epub 2019 May 11.

Institut für Chemie, Technische Universität Berlin, Müller-Breslau-Straße 10, 10623 Berlin, Germany.

Engineering aminoacyl-tRNA synthetases (aaRSs) provides access to the ribosomal incorporation of noncanonical amino acids via genetic code expansion. Conventional targeted mutagenesis libraries with 5-7 positions randomized cover only marginal fractions of the vast sequence space formed by up to 30 active site residues. This frequently results in selection of weakly active enzymes. To overcome this limitation, we use computational enzyme design to generate a focused library of aaRS variants. For aaRS enzyme redesign, photocaged -nitrobenzyl tyrosine (ONBY) was chosen as substrate due to commercial availability and its diverse applications. Diversifying 17 first- and second-shell sites and performing conventional aaRS positive and negative selection resulted in a high-activity aaRS. This TyrRS variant carries ten mutations and outperforms previously reported ONBY-specific aaRS variants isolated from traditional libraries. In response to a single in-frame amber stop codon, it mediates the in vivo incorporation of ONBY with an efficiency matching that of the wild type TyrRS enzyme acylating cognate tyrosine. These results exemplify an improved general strategy for aaRS library design and engineering.
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http://dx.doi.org/10.3390/ijms20092343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6539999PMC
May 2019

In-Cell Synthesis of Bioorthogonal Alkene Tag S-Allyl-Homocysteine and Its Coupling with Reprogrammed Translation.

Int J Mol Sci 2019 May 9;20(9). Epub 2019 May 9.

Institut für Chemie, Technische Universität Berlin, Müller-Breslau-Str. 10, D-10623 Berlin, Germany.

In this study, we report our initial results on in situ biosynthesis of S-allyl-l-homocysteine (Sahc) by simple metabolic conversion of allyl mercaptan in , which served as the host organism endowed with a direct sulfhydration pathway. The intracellular synthesis we describe in this study is coupled with the direct incorporation of Sahc into proteins in response to methionine codons. Together with -acetyl-homoserine, allyl mercaptan was added to the growth medium, followed by uptake and intracellular reaction to give Sahc. Our protocol efficiently combined the in vivo synthesis of Sahc via metabolic engineering with reprogrammed translation, without the need for a major change in the protein biosynthesis machinery. Although the system needs further optimisation to achieve greater intracellular Sahc production for complete protein labelling, we demonstrated its functional versatility for photo-induced thiol-ene coupling and the recently developed phosphonamidate conjugation reaction. Importantly, deprotection of Sahc leads to homocysteine-containing proteins-a potentially useful approach for the selective labelling of thiols with high relevance in various medical settings.
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http://dx.doi.org/10.3390/ijms20092299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6539321PMC
May 2019

Expanding the DOPA Universe with Genetically Encoded, Mussel-Inspired Bioadhesives for Material Sciences and Medicine.

Chembiochem 2019 09 24;20(17):2163-2190. Epub 2019 Jun 24.

Institute of Chemistry, Technical University of Berlin, Müller-Breslau-Strasse 10, Berlin, 10623, Germany.

Catechols are a biologically relevant group of aromatic diols that have attracted much attention as mediators of adhesion of "bio-glue" proteins in mussels of the genus Mytilus. These organisms use catechols in the form of the noncanonical amino acid l-3,4-dihydroxyphenylalanine (DOPA) as a building block for adhesion proteins. The DOPA is generated post-translationally from tyrosine. Herein, we review the properties, natural occurrence, and reactivity of catechols in the design of bioinspired materials. We also provide a basic description of the mussel's attachment apparatus, the interplay between its different molecules that play a crucial role in adhesion, and the role of post-translational modifications (PTMs) of these proteins. Our focus is on the microbial production of mussel foot proteins with the aid of orthogonal translation systems (OTSs) and the use of genetic code engineering to solve some fundamental problems in the bioproduction of these bioadhesives and to expand their chemical space. The major limitation of bacterial expression systems is their intrinsic inability to introduce PTMs. OTSs have the potential to overcome these challenges by replacing canonical amino acids with noncanonical ones. In this way, PTM steps are circumvented while the genetically programmed precision of protein sequences is preserved. In addition, OTSs should enable spatiotemporal control over the complex adhesion process, because the catechol function can be masked by suitable chemical protection. Such caged residues can then be noninvasively unmasked by, for example, UV irradiation or thermal treatment. All of these features make OTSs based on genetic code engineering in reprogrammed microbial strains new and promising tools in bioinspired materials science.
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http://dx.doi.org/10.1002/cbic.201900030DOI Listing
September 2019

Promotion of the collagen triple helix in a hydrophobic environment.

Org Biomol Chem 2019 02;17(9):2502-2507

Institute of Chemistry, Technical University of Berlin, Müller-Breslau-Str. 10, Berlin, 10623, Germany.

In contrast to many other water-soluble peptide arrangements, the formation of a triple helix in collagen proceeds inside out: polar glycyl residues form the interior, whereas nonpolar prolyl side chains constitute the exterior. In our work, we decided to exploit this aspect of the peptide architecture in order to create hyperstable collagen mimicking peptides (CMPs). The key element of this study is the environment. Given that the peptide assembles in a nonpolar medium, the collapse of the polar peptide backbone into the triple helix should become more favorable. Following this idea, we prepared CMPs based on hydrophobic proline analogues. The synthesis was performed by a combination of liquid- and solid-phase approaches: first, hexapeptides were prepared in solution, and then these were launched into conventional Fmoc-based peptide synthesis on a solid support. The resulting peptides showed an excellent signal of the triple helix in the model nonpolar solvent (octanol) according to circular dichroism observations. In a study of a series of oligomers, we found that the minimal length of the peptides required for triple helical assembly is substantially lower compared to water-soluble CMPs. Our results suggest further explorations of the CMPs in hydrophobic media; in particular, we highlight the suggestion that collagen could be converted into a membrane protein.
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http://dx.doi.org/10.1039/c9ob00070dDOI Listing
February 2019

Alternative Biochemistries for Alien Life: Basic Concepts and Requirements for the Design of a Robust Biocontainment System in Genetic Isolation.

Genes (Basel) 2018 Dec 28;10(1). Epub 2018 Dec 28.

Institut für Chemie, Technische Universität Berlin Müller-Breslau-Straße 10, 10623 Berlin, Germany.

The universal genetic code, which is the foundation of cellular organization for almost all organisms, has fostered the exchange of genetic information from very different paths of evolution. The result of this communication network of potentially beneficial traits can be observed as modern biodiversity. Today, the genetic modification techniques of synthetic biology allow for the design of specialized organisms and their employment as tools, creating an artificial biodiversity based on the same universal genetic code. As there is no natural barrier towards the proliferation of genetic information which confers an advantage for a certain species, the naturally evolved genetic pool could be irreversibly altered if modified genetic information is exchanged. We argue that an alien genetic code which is incompatible with nature is likely to assure the inhibition of all mechanisms of genetic information transfer in an open environment. The two conceivable routes to synthetic life are either de novo cellular design or the successive alienation of a complex biological organism through laboratory evolution. Here, we present the strategies that have been utilized to fundamentally alter the genetic code in its decoding rules or its molecular representation and anticipate future avenues in the pursuit of robust biocontainment.
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http://dx.doi.org/10.3390/genes10010017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356944PMC
December 2018

Site-Specific Chemoselective Pyrrolysine Analogues Incorporation Using the Cell-Free Protein Synthesis System.

ACS Synth Biol 2019 02 28;8(2):381-390. Epub 2019 Jan 28.

biotechrabbit GmbH , 12489 Berlin , Germany.

Cell-free protein synthesis (CFPS) is a fast and convenient way to synthesize proteins for analytical studies and applications. CFPS, when equipped with a suitable orthogonal pair, allows for protein-site-directed labeling with desired functionalities such as fluorescent dyes or therapeutic groups that are needed to tailor proteins for analytical applications. In this context, chemoselective reactive pyrrolysine analogues (CR-OAs) are of particular value, as this class of unnatural amino acids, among other useful properties, covers a wide range of different chemoselective reactions. In this study, we present a flexible approach that facilitates incorporation of CR-OAs in CFPS systems. In particular, a fairly simple addition of two expression plasmids in our cell-free system, one encoding pyrrolysyl-tRNA synthetase and the other one the target protein, enabled ribosomal synthesis of proteins in the half-milligram range with the pre-installed orthogonal reactivity, easily modifiable by using mild, copper-free bioorthogonal chemistry. Our CFPS system allows rapid and highly customizable expression, as shown by several examples of successful site-directed fluorescence labeling. The feasibility of our CFPS system for protein analytics is further proved by demonstrating the functional integrity of a labeled protein by interaction measurements using microscale thermophoresis.
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http://dx.doi.org/10.1021/acssynbio.8b00421DOI Listing
February 2019

Site-Resolved Observation of Vibrational Energy Transfer Using a Genetically Encoded Ultrafast Heater.

Angew Chem Int Ed Engl 2019 02 6;58(9):2899-2903. Epub 2019 Feb 6.

Institut für Chemie, Technische Universität Berlin, Müller-Breslau-Str. 10, 10623, Berlin, Germany.

Allosteric information transfer in proteins has been linked to distinct vibrational energy transfer (VET) pathways in a number of theoretical studies. Experimental evidence for such pathways, however, is sparse because site-selective injection of vibrational energy into a protein, that is, localized heating, is required for their investigation. Here, we solved this problem by the site-specific incorporation of the non-canonical amino acid β-(1-azulenyl)-l-alanine (AzAla) through genetic code expansion. As an exception to Kasha's rule, AzAla undergoes ultrafast internal conversion and heating after S excitation while upon S excitation, it serves as a fluorescent label. We equipped PDZ3, a protein interaction domain of postsynaptic density protein 95, with this ultrafast heater at two distinct positions. We indeed observed VET from the incorporation sites in the protein to a bound peptide ligand on the picosecond timescale by ultrafast IR spectroscopy. This approach based on genetically encoded AzAla paves the way for detailed studies of VET and its role in a wide range of proteins.
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http://dx.doi.org/10.1002/anie.201812995DOI Listing
February 2019

Long-Range Modulations of Electric Fields in Proteins.

J Phys Chem B 2018 09 28;122(35):8330-8342. Epub 2018 Aug 28.

Institut für Chemie , Technische Universität Berlin , Sekr. PC14, Straße des 17. Juni 135 , D-10623 Berlin , Germany.

Electrostatic interactions are essential for controlling the protein structure and function. Whereas so far experimental and theoretical efforts focused on the effect of local electrostatics, this work aims at elucidating the long-range modulation of electric fields in proteins upon binding to charged surfaces. The study is based on cytochrome c (Cytc) variants carrying nitrile reporters for the vibrational Stark effect that are incorporated into the protein via genetic engineering and chemical modification. The Cytc variants were thoroughly characterized with respect to possible structural perturbations due to labeling. For the proteins in solution, the relative hydrogen bond occupancy and the calculated electric fields, both obtained from molecular dynamics (MD) simulations, and the experimental nitrile stretching frequencies were used to develop a relationship for separating hydrogen-bonding and non-hydrogen-bonding electric field effects. This relationship provides an excellent description for the stable Cytc variants in solution. For the proteins bound to Au electrodes coated with charged self-assembled monolayers (SAMs), the underlying MD simulations can only account for the electric field changes Δ E due to the formation of the electrostatic SAM-Cytc complexes but not for the additional contribution, Δ E, representing the consequences of the potential drops over the electrode/SAM/protein interfaces. Both Δ E and Δ E, determined at distances between 20 and 30 Å with respect to the SAM surface, are comparable in magnitude to the non-hydrogen-bonding electric field in the unbound protein. This long-range modulation of the internal electric field may be of functional relevance for proteins in complexes with partner proteins (Δ E) and attached to membranes (Δ E + Δ E).
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http://dx.doi.org/10.1021/acs.jpcb.8b03870DOI Listing
September 2018

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

J Vis Exp 2018 05 4(135). Epub 2018 May 4.

Department of Biocatalysis, Institute of Chemistry, Technische Universität Berlin.

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all variations are based on the 20 canonical amino acids (cAAs). As a way to introduce additional physicochemical properties into polypeptides, the incorporation of non-canonical amino acids (ncAAs) is increasingly used in protein engineering. Due to their relatively short length, the modification of ribosomally synthesized and post-translationally modified peptides by ncAAs is particularly attractive. New functionalities and chemical handles can be generated by specific modifications of individual residues. The selective pressure incorporation (SPI) method utilizes auxotrophic host strains that are deprived of an essential amino acid in chemically defined growth media. Several structurally and chemically similar amino acid analogs can then be activated by the corresponding aminoacyl-tRNA synthetase and provide residue-specific cAA(s) → ncAA(s) substitutions in the target peptide or protein sequence. Although, in the context of the SPI method, ncAAs are also incorporated into the host proteome during the phase of recombinant gene expression, the majority of the cell's resources are assigned to the expression of the target gene. This enables efficient residue-specific incorporation of ncAAs often accompanied with high amounts of modified target. The presented work describes the in vivo incorporation of six proline analogs into the antimicrobial peptide nisin, a lantibiotic naturally produced by Lactococcus lactis. Antimicrobial properties of nisin can be changed and further expanded during its fermentation and expression in auxotrophic Escherichia coli strains in defined growth media. Thereby, the effects of residue-specific replacement of cAAs with ncAAs can deliver changes in antimicrobial activity and specificity. Antimicrobial activity assays and fluorescence microscopy are used to test the new nisin variants for growth inhibition of a Gram-positive Lactococcus lactis indicator strain. Mass spectroscopy is used to confirm ncAA incorporation in bioactive nisin variants.
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http://dx.doi.org/10.3791/57551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6101111PMC
May 2018

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

J Vis Exp 2018 04 27(134). Epub 2018 Apr 27.

Institute of Chemistry L 1, Department of Biocatalysis, Technical University of Berlin.

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.
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http://dx.doi.org/10.3791/57017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6100899PMC
April 2018

Painting argyrins blue: Negishi cross-coupling for synthesis of deep-blue tryptophan analogue β-(1-azulenyl)-l alanine and its incorporation into argyrin C.

Bioorg Med Chem 2018 10 26;26(19):5259-5269. Epub 2018 Mar 26.

Institute for Organic Chemistry, Leibniz University Hannover, Schneiderberg 1B, 30167 Hannover and Centre of Biomolecular Drug Research (BMWZ), Schneiderberg 38, 30167 Hannover, Germany; Helmholtz Centre for Infection Research GmbH (HZI), Inhoffenstraße 7, 38124 Braunschweig, Germany. Electronic address:

The argyrins are a family of non-ribosomal peptides that exhibits different biological activities through only small structural changes. Ideally, a biologically active molecule can be tracked and observed in a variety of biological and clinical settings in a non-invasive manner. As a step towards this goal, we report here a chemical synthesis of unnatural deep blue amino acid β-(1-azulenyl)-l alanine with different fluorescence and photophysical properties, which allows a spectral separation from the native tryptophan signal. This might be especially useful for cell localization studies and visualizing the targeted proteins. In particular, the synthesis of β-(1-azulenyl)-l alanine was achieved through a Negishi coupling which proved to be a powerful tool for the synthesis of unnatural tryptophan analogs. Upon β-(1-azulenyl)-l alanine incorporation into argyrin C, deep blue octapeptide variant was spectrally and structurally characterized.
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http://dx.doi.org/10.1016/j.bmc.2018.03.037DOI Listing
October 2018

Expanding the Genetic Code of and to Incorporate Non-canonical Amino Acids for Production of Modified Lantibiotics.

Front Microbiol 2018 6;9:657. Epub 2018 Apr 6.

Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, Netherlands.

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.
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http://dx.doi.org/10.3389/fmicb.2018.00657DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5897534PMC
April 2018

Transmembrane Polyproline Helix.

J Phys Chem Lett 2018 May 13;9(9):2170-2174. Epub 2018 Apr 13.

Institute of Chemistry , Technical University of Berlin , Müller-Breslau-Strasse 10 , Berlin 10623 , Germany.

The third most abundant polypeptide conformation in nature, the polyproline-II helix, is a polar, extended secondary structure with a local organization stabilized by intercarbonyl interactions within the peptide chain. Here we design a hydrophobic polyproline-II helical peptide based on an oligomeric octahydroindole-2-carboxylic acid scaffold and demonstrate its transmembrane alignment in model lipid bilayers by means of solid-state F NMR. As result, we provide a first example of a purely artificial transmembrane peptide with a structural organization that is not based on hydrogen-bonding.
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http://dx.doi.org/10.1021/acs.jpclett.8b00829DOI Listing
May 2018

Exploring hydrophobicity limits of polyproline helix with oligomeric octahydroindole-2-carboxylic acid.

J Pept Sci 2018 Jun 27;24(4-5):e3076. Epub 2018 Mar 27.

Institute of Chemistry, Technical University of Berlin, Müller-Breslau-Str. 10, Berlin, 10623, Germany.

The polyproline-II helix is the most extended naturally occurring helical structure and is widely present in polar, exposed stretches and "unstructured" denatured regions of polypeptides. Can it be hydrophobic? In this study, we address this question using oligomeric peptides formed by a hydrophobic proline analogue, (2S,3aS,7aS)-octahydroindole-2-carboxylic acid (Oic). Previously, we found the molecular principles underlying the structural stability of the polyproline-II conformation in these oligomers, whereas the hydrophobicity of the peptide constructs remains to be examined. Therefore, we investigated the octan-1-ol/water partitioning and inclusion in detergent micelles of the oligo-Oic peptides. The results showed that the hydrophobicity is remarkably enhanced in longer oligomeric sequences, and the oligo-Oic peptides with 3 to 4 residues and higher are specific towards hydrophobic environments. This contrasts significantly to the parent oligoproline peptides, which were moderately hydrophilic. With these findings, we have demonstrated that the polyproline-II structure is compatible with nonpolar media, whereas additional manipulations of the terminal functionalities feature solubility in extremely nonpolar solvents such as hexane.
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http://dx.doi.org/10.1002/psc.3076DOI Listing
June 2018

Self-Directed in Cell Production of Methionine Analogue Azidohomoalanine by Synthetic Metabolism and Its Incorporation into Model Proteins.

Methods Mol Biol 2018 ;1728:127-135

Biocatalysis Group, Department of Chemistry, Berlin Institute of Technology/TU Berlin, Berlin, Germany.

Common protocols for the incorporation of noncanonical amino acids (ncAAs) into proteins require addition of the desired ncAA to the growth medium, its cellular uptake, and subsequent intracellular accumulation. This feeding scheme is generally suitable for small-scale proof-of-concept incorporation experiments. However, it is no general solution for orthogonal translation of ncAAs, as their chemical synthesis is generally tedious and expensive. Here, we describe a simple protocol that efficiently couples in situ semi-synthetic biosynthesis of L-azidohomoalanine and its incorporation into proteins at L-methionine (Met) positions. In our metabolically engineered Met-auxotrophic Escherichia coli strain, Aha is biosynthesized from externally added sodium azide and O-acetyl-L-homoserine as inexpensive precursors. This represents an efficient platform for expression of azide-containing proteins suitable for site-selective bioorthogonal strategies aimed at noninvasive protein modifications (Tornøe et al., J Org Chem 67:3057-3064, 2002; Kiick et al., Angew Chem Int Ed 39:2148-2152, 2000; Budisa, Angew Chem Int Ed Engl 47:6426-6463, 2004; van Hest, J Am Chem Soc 122:1282-1288, 2000).
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http://dx.doi.org/10.1007/978-1-4939-7574-7_7DOI Listing
December 2018

Hydrolysis, polarity, and conformational impact of C-terminal partially fluorinated ethyl esters in peptide models.

Beilstein J Org Chem 2017 16;13:2442-2457. Epub 2017 Nov 16.

Biocatalysis group, Institute of Chemistry, Technical University of Berlin, Müller-Breslau-Strasse 10, Berlin 10623, Germany.

Fluorinated moieties are highly valuable to chemists due to the sensitive NMR detectability of the F nucleus. Fluorination of molecular scaffolds can also selectively influence a molecule's polarity, conformational preferences and chemical reactivity, properties that can be exploited for various chemical applications. A powerful route for incorporating fluorine atoms in biomolecules is last-stage fluorination of peptide scaffolds. One of these methods involves esterification of the C-terminus of peptides using a diazomethane species. Here, we provide an investigation of the physicochemical consequences of peptide esterification with partially fluorinated ethyl groups. Derivatives of -acetylproline are used to model the effects of fluorination on the lipophilicity, hydrolytic stability and on conformational properties. The conformational impact of the 2,2-difluoromethyl ester on several neutral and charged oligopeptides was also investigated. Our results demonstrate that partially fluorinated esters undergo variable hydrolysis in biologically relevant buffers. The hydrolytic stability can be tailored over a broad pH range by varying the number of fluorine atoms in the ester moiety or by introducing adjacent charges in the peptide sequence.
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http://dx.doi.org/10.3762/bjoc.13.241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5704756PMC
November 2017