Imidazole-amino acids. Conformational switch under tautomer and pH change.

Replacement of the main chain peptide bond by imidazole ring seems to be a promising tool for the peptide-based drug design, due to the specific prototropic tautomeric as well as amphoteric properties. In this study, we present that both tautomer and pH change can cause a conformational switch of the studied residues of alanine (1-4) and dehydroalanine (5-8) with the C-terminal peptide group replaced by imidazole. The DFT methods are applied and an environment of increasing polarity is simulated. The conformational maps (Ramachandram diagrams) are presented and the stability of possible conformations is discussed. The neutral forms, tautomers τ (1) and π (2), adapt the conformations αRτ (φ, ψ = - 75°, - 114°) and C7eq (φ, ψ = - 75°, 66°), respectively. Their torsion angles ψ differ by about 180°, which results in a considerable impact on the peptide chain conformation. The cation form (3) adapts both these conformations, whereas the anion analogue (4) prefers the conformations C5 (φ, ψ = - 165°, - 178°) and ß2 (φ, ψ ~ - 165°, - 3°). Dehydroamino acid analogues, the tautomers τ (5) and π (6) as well as the anion form (8), have a strong tendency toward the conformations ß2 (φ, ψ = - 179°, 0°) and C5 (φ, ψ = - 180°, 180°). The preferences of the protonated imidazolium form (7) depend on the environment. The imidazole ring, acting as a donor or acceptor of the hydrogen bonds created within the studied residues, has a profound effect on the type of conformation.

Chemoselective restoration of para-azido-phenylalanine at multiple sites in proteins.

The site-specific incorporation of nonstandard amino acids (nsAAs) during translation has expanded the chemistry and function of proteins. The nsAA para-azido-phenylalanine (pAzF) encodes a biorthogonal chemical moiety that facilitates "click" reactions to attach diverse chemical groups for protein functionalization. However, the azide moiety is unstable in physiological conditions and is reduced to para-amino-phenylalanine (pAF). Azide reduction decreases the yield of pAzF residues in proteins to 50%-60% per azide and limits protein functionalization by click reactions. Here, we describe the use of a pH-tunable diazotransfer reaction that converts pAF to pAzF at >95% efficiency in proteins. The method selectively restores pAzF at multiple sites per protein without introducing off-target modifications. This work addresses a key limitation in the production of pAzF-containing proteins by restoring azides for multi-site functionalization with diverse chemical moieties, setting the stage for the production of genetically encoded biomaterials with broad applications in biotherapeutics, materials science, and biotechnology.

A Computer-Based Methodology to Design Non-Standard Peptides Potentially Able to Prevent HOX-PBX1-Associated Cancer Diseases.

In the last decades, HOX proteins have been extensively studied due to their pivotal role in transcriptional events. HOX proteins execute their activity by exploiting a cooperative binding to PBX proteins and DNA. Therefore, an increase or decrease in HOX activity has been associated with both solid and haematological cancer diseases. Thus, inhibiting HOX-PBX interaction represents a potential strategy to prevent these malignancies, as demonstrated by the patented peptide HTL001 that is being studied in clinical trials. In this work, a computational study is described to identify novel potential peptides designed by employing a database of non-natural amino acids. For this purpose, residue scanning of the HOX minimal active sequence was performed to select the mutations to be further processed. According to these results, the peptides were point-mutated and used for Molecular Dynamics (MD) simulations in complex with PBX1 protein and DNA to evaluate complex binding stability. MM-GBSA calculations of the resulting MD trajectories were exploited to guide the selection of the most promising mutations that were exploited to generate twelve combinatorial peptides. Finally, the latter peptides in complex with PBX1 protein and DNA were exploited to run MD simulations and the ΔGbinding average values of the complexes were calculated. Thus, the analysis of the results highlighted eleven combinatorial peptides that will be considered for further assays.

Thiazole-amino acids: influence of thiazole ring on conformational properties of amino acid residues.

Post-translational modified thiazole-amino acid (Xaa-Tzl) residues have been found in macrocyclic peptides (e.g., thiopeptides and cyanobactins), which mostly inhibit protein synthesis in Gram + bacteria. Conformational study of the series of model compounds containing this structural motif with alanine, dehydroalanine, dehydrobutyrine and dehydrophenylalanine were performed using DFT method in various environments. The solid-state crystal structure conformations of thiazole-amino acid residues retrieved from the Cambridge Structural Database were also analysed. The studied structural units tend to adopt the unique semi-extended ß2 conformation; which is stabilised mainly by N-H⋯NTzl hydrogen bond, and for dehydroamino acids also by π-electron conjugation. The conformational preferences of amino acids with a thiazole ring were compared with oxazole analogues and the role of the sulfur atom in stabilising the conformations of studied peptides was discussed.

Advances in engineering microbial biosynthesis of aromatic compounds and related compounds.

Aromatic compounds have broad applications and have been the target of biosynthetic processes for several decades. New biomolecular engineering strategies have been applied to improve production of aromatic compounds in recent years, some of which are expected to set the stage for the next wave of innovations. Here, we will briefly complement existing reviews on microbial production of aromatic compounds by focusing on a few recent trends where considerable work has been performed in the last 5 years. The trends we highlight are pathway modularization and compartmentalization, microbial co-culturing, non-traditional host engineering, aromatic polymer feedstock utilization, engineered ring cleavage, aldehyde stabilization, and biosynthesis of non-standard amino acids. Throughout this review article, we will also touch on unmet opportunities that future research could address.

Incorporation of non-standard amino acids into proteins: challenges, recent achievements, and emerging applications.

The natural genetic code only allows for 20 standard amino acids in protein translation, but genetic code reprogramming enables the incorporation of non-standard amino acids (NSAAs). Proteins containing NSAAs provide enhanced or novel properties and open diverse applications. With increased attention to the recent advancements in synthetic biology, various improved and novel methods have been developed to incorporate single and multiple distinct NSAAs into proteins. However, various challenges remain in regard to NSAA incorporation, such as low yield and misincorporation. In this review, we summarize the recent efforts to improve NSAA incorporation by utilizing orthogonal translational system optimization, cell-free protein synthesis, genomically recoded organisms, artificial codon boxes, quadruplet codons, and orthogonal ribosomes, before closing with a discussion of the emerging applications of NSAA incorporation.

Cotranslational incorporation of non-standard amino acids using cell-free protein synthesis.

Over the last years protein engineering using non-standard amino acids has gained increasing attention. As a result, improved methods are now available, enabling the efficient and directed cotranslational incorporation of various non-standard amino acids to equip proteins with desired characteristics. In this context, the utilization of cell-free protein synthesis is particularly useful due to the direct accessibility of the translational machinery and synthesized proteins without having to maintain a vital cellular host. We review prominent methods for the incorporation of non-standard amino acids into proteins using cell-free protein synthesis. Furthermore, a list of non-standard amino acids that have been successfully incorporated into proteins in cell-free systems together with selected applications is provided.

Improving cell-free protein synthesis through genome engineering of Escherichia coli lacking release factor 1.

Site-specific incorporation of non-standard amino acids (NSAAs) into proteins opens the way to novel biological insights and applications in biotechnology. Here, we describe the development of a high yielding cell-free protein synthesis (CFPS) platform for NSAA incorporation from crude extracts of genomically recoded Escherichia coli lacking release factor 1. We used genome engineering to construct synthetic organisms that, upon cell lysis, lead to improved extract performance. We targeted five potential negative effectors to be disabled: the nuclease genes rna, rnb, csdA, mazF, and endA. Using our most productive extract from strain MCJ.559 (csdA(-) endA(-)), we synthesized 550±40 µg mL(-1) of modified superfolder green fluorescent protein containing p-acetyl-L-phenylalanine. This yield was increased to ∼1300 µg mL(-1) when using a semicontinuous method. Our work has implications for using whole genome editing for CFPS strain development, expanding the chemistry of biological systems, and cell-free synthetic biology.

Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis.

Incorporating non-standard amino acids (NSAAs) into proteins enables new chemical properties, new structures, and new functions. In recent years, improvements in cell-free protein synthesis (CFPS) systems have opened the way to accurate and efficient incorporation of NSAAs into proteins. The driving force behind this development has been three-fold. First, a technical renaissance has enabled high-yielding (>1 g/L) and long-lasting (>10 h in batch operation) CFPS in systems derived from Escherichia coli. Second, the efficiency of orthogonal translation systems (OTSs) has improved. Third, the open nature of the CFPS platform has brought about an unprecedented level of control and freedom of design. Here, we review recent developments in CFPS platforms designed to precisely incorporate NSAAs. In the coming years, we anticipate that CFPS systems will impact efforts to elucidate structure/function relationships of proteins and to make biomaterials and sequence-defined biopolymers for medical and industrial applications.