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Unnatural amino acid

Heinz Neumann, Petra Neumann-Staubitz, Anna Witte, Daniel Summerer
The genetic incorporation of unnatural amino acids (UAAs) into proteins by amber suppression technology provides unique avenues to study protein structure, function and interactions both in vitro and in living cells and organisms. This approach has been particularly useful for studying mechanisms of epigenetic chromatin regulation, since these extensively involve dynamic changes in structure, complex formation and posttranslational modifications that are difficult to access by traditional approaches. Here, we review recent achievements in this field, emphasizing UAAs that help to unravel protein-protein interactions in cells by photo-crosslinking or that allow the biosynthesis of proteins with defined posttranslational modifications for studying their function and turnover in vitro and in cells...
February 13, 2018: Current Opinion in Chemical Biology
Bertrand Jordan
Alternative bases that can fit into the DNA double helix have now been used in vivo to direct the synthesis of proteins incorporating unnatural amino acids. This bioengineering feat is significant at both the conceptual and the practical levels.
February 2018: Médecine Sciences: M/S
Jia Ying Cheang, Peter Michael Moyle
Glucagon-like peptide-1 (GLP-1) is secreted by intestinal L-cells following food intake, and plays an important role in glucose homeostasis due to its stimulation of glucose-dependent insulin secretion. Further, GLP-1 is also associated with protective effects on pancreatic β-cells and the cardiovascular system, reduced appetite, and weight loss, making GLP-1 derivatives an exciting treatment for type 2 diabetes and obesity. Despite these benefits, wild-type GLP-1 exhibits a short circulation time, due to its poor metabolic stability and rapid renal clearance, and needs to be administered by injection, making it a poor therapeutic agent...
February 11, 2018: ChemMedChem
Makan Khoshnejad, Colin Fred Greineder, Katherine W Pulsipher, Carlos H Villa, Burcin Altun, Daniel C Pan, Andrew Tsourkas, Ivan J Dmochowski, Vladimir Muzykantov
Genetic incorporation of biologically orthogonal functional groups into macromolecules has the potential to yield efficient, controlled, reproducible, site-specific conjugation of affinity ligands, contrast agents, or therapeutic cargoes. Here, we applied this approach to ferritin, a ubiquitous iron-storage protein that self-assembles into multimeric nanocages with remarkable stability, size uniformity (12 nm), and endogenous capacity for loading and transport of a variety of inorganic and organic cargoes. The unnatural amino acid, 4-azidophenylalanine (4-AzF), was incorporated at different sites in the human ferritin light chain (hFTL) to allow site-specific conjugation of alkyne-containing small molecules or affinity ligands to the exterior surface of the nanocage...
February 10, 2018: Bioconjugate Chemistry
J Hernandez-Montelongo, Y R Corrales Ureña, D Machado, M Lancelloti, M P Pinheiro, K Rischka, P N Lisboa-Filho, M A Cotta
Staphylococcus epidermidis is a gram-positive bacterium, and one of the most prevalent causes of nosocomial infections due to its strong ability to form biofilms on catheters and surgical implants. Here we explore the antimicrobial properties of Tet-124 peptides, which are part of the innate defense against different multicellular organisms in nature. Two different Tet-124 peptides were immobilized on a polyethylenimine (PEI) film to determine their impact on the antimicrobial properties: KLWWMIRRW (Tet-124), which contains only natural amino acids, and KLWWMIRRWG-(F-Br)-G (F-Br = 4-Bromophenylalanine), a modified Tet-124 sequence with the addition of an unnatural amino acid...
February 5, 2018: Colloids and Surfaces. B, Biointerfaces
Lloyd Davis, Sebastian Greiss
Site-specific incorporation of unnatural amino acids (UAAs) has greatly expanded the toolkit available to study biological phenomena in single cells. However, to address questions involving complex cellular interactions such as development, ageing, and the functions of the nervous system it is often necessary to use multicellular model organisms. The nematode Caenorhabditis elegans was the first organism to have its genetic code expanded. Due to its small size, ease of cultivation, and excellent UAA incorporation efficiency, C...
2018: Methods in Molecular Biology
Rachel E Kelemen, Sarah B Erickson, Abhishek Chatterjee
The ability to modify the capsid proteins of human viruses is desirable both for installing probes to study their structure and function, and to attach retargeting agents to engineer viral infectivity. However, the installation of such capsid modifications currently faces two major challenges: (1) The complex and delicate capsid proteins often do not tolerate large modifications, and (2) capsid proteins are composed of the 20 canonical amino acids, precluding site-specific chemical modification of the virus...
2018: Methods in Molecular Biology
Ji-Yong Kang, Daichi Kawaguchi, Lei Wang
Deciphering neuronal networks governing specific brain functions is a longstanding mission in neuroscience, yet global manipulation of protein functions pharmacologically or genetically lacks sufficient specificity to reveal a neuronal protein's function in a particular neuron or a circuitry. Photostimulation presents a great venue for researchers to control neuronal proteins with high temporal and spatial resolution. Recently, an approach to optically control the function of a neuronal protein directly in neurons has been demonstrated using genetically encoded light-sensitive Unnatural amino acids (Uaas)...
2018: Methods in Molecular Biology
Christian Hoffmann, Heinz Neumann, Petra Neumann-Staubitz
The installation of unnatural amino acids into proteins of living cells is an enabling technology that facilitates an enormous number of applications. UV-activatable crosslinker amino acids allow the formation of a covalent bond between interaction partners in living cells with nearly perfect spatial and temporal control. Here, we describe how this method can be employed to map chromatin interactions and to follow these interactions across the cell cycle in synchronized yeast populations. This method thereby provides unprecedented insights into the molecular events controlling chromatin reorganization in mitosis...
2018: Methods in Molecular Biology
Jingxuan He, Charles E Melançon
Site-specific, genetic incorporation of unnatural amino acids (UAAs) into proteins in living cells using engineered orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs is a powerful tool for studying and manipulating protein structure and function. To date, UAA incorporation systems have been developed for several bacterial and eukaryotic model hosts. Due to the importance of Streptomyces as prolific producers of bioactive natural products and as model hosts for natural product biosynthesis and bioengineering studies, we have developed systems for the incorporation of the UAAs p-iodo-L-phenylalanine (pIPhe) and p-azido-L-phenylalanine (pAzPhe) into green fluorescent protein (GFP) in Streptomyces venezuelae ATCC 15439...
2018: Methods in Molecular Biology
Jonas Helma, Heinrich Leonhardt, Christian P R Hackenberger, Dominik Schumacher
Tub-tag labeling is a chemoenzymatic method that enables the site-specific labeling of proteins. Here, the natural enzyme tubulin tyrosine ligase incorporates noncanonical tyrosine derivatives to the terminal carboxylic acid of proteins containing a 14-amino acid recognition sequence called Tub-tag. The tyrosine derivative carries a unique chemical reporter allowing for a subsequent bioorthogonal modification of proteins with a great variety of probes. Here, we describe the Tub-tag protein modification protocol in detail and explain its utilization to generate labeled proteins for advanced applications in cell biology, imaging, and diagnostics...
2018: Methods in Molecular Biology
Karel Mena-Ulecia, Fabian Gonzalez-Norambuena, Ariela Vergara-Jaque, Horacio Poblete, William Tiznado, Julio Caballero
Protein kinases (PKs) discriminate between closely related sequences that contain serine, threonine, and/or tyrosine residues. Such specificity is defined by the amino acid sequence surrounding the phosphorylatable residue, so that it is possible to identify an optimal recognition motif (ORM) for each PK. The ORM for the protein kinase A (PKA), a well-known member of the PK family, is the sequence RRX(S/T)X, where arginines at the -3 and -2 positions play a key role with respect to the primed phosphorylation site...
February 5, 2018: Journal of Computational Chemistry
Hannes Ludewig, Clarissa M Czekster, Emilia Oueis, Elizabeth S Munday, Mohammed Arshad, Silvia A Synowsky, Andrew F Bent, James H Naismith
Cyclic ribosomally derived peptides possess diverse bioactivities and are currently of major interest in drug development. However, it can be chemically challenging to synthesize these molecules, hindering the diversification and testing of cyclic peptide leads. Enzymes used in vitro offer a solution to this, however peptide macrocyclization remains the bottleneck. PCY1, involved in the biosynthesis of plant orbitides, belongs to the class of prolyl oligopeptidases and natively displays substrate promiscuity...
January 29, 2018: ACS Chemical Biology
Wenjun Li, Xianhong Xu, Yang Liu, Hua Gao, Yuyu Cheng, Pengfei Li
This work describes the first enantioselective 1,6-additions of azlactones to para-quinone methides. In the presence of a chiral phosphoric acid, 1,6-adducts were obtained in high yields (up to 96%) with excellent diastereoselectivities and enantioselectivities (all >20:1 diastereoselectivity ratio (dr), up to 99% enantiomeric excess (ee)). Importantly, the method offers a facile synthetic approach, not only to enantiopure α,α-disubstituted α-amino acid esters, but also to unnatural enantioenriched β,β-diaryl-α-amino acid esters bearing adjacent tertiary and quaternary stereogenic centers...
January 24, 2018: Organic Letters
Avisek Ghose, Mariana Amaro, Petr Kovaricek, Martin Hof, Jan Sykora
Coumarin derivatives are well known fluorescence reporters for investigating biological systems due to their strong micro-environment sensitivity. Despite of having wide range of environment sensitive fluorescence probes, the potential of 6,7-dimethoxy-coumarin has not been studied extensively so far. With a perspective of its use in protein studies, namely using the unnatural amino acid technology or as a substrate for hydrolase enzymes, we study acetyloxymethyl-6,7-dimethoxycoumarin (Ac-DMC). We investigate the photophysics and hydration dynamics of this dye in aerosol-OT (AOT) reverse micelles at various water contents using the time dependent fluorescence shift (TDFS) method...
January 23, 2018: Methods and Applications in Fluorescence
An De Prins, Charlotte Martin, Yannick Van Wanseele, Louise Julie Skov, Csaba Tömböly, Dirk Tourwé, Vicky Caveliers, Ann Van Eeckhaut, Birgitte Holst, Mette Marie Rosenkilde, Ilse Smolders, Steven Ballet
Neuromedin U (NMU) is a highly conserved endogenous peptide that is involved in a wide range of physiological processes such as regulation of feeding behavior, the stress response and nociception. The major limitation to use NMU as a therapeutic is its short half-life. Here, we describe the development of a set of novel NMU-analogs based on NMU-8, by introducing unnatural amino acids into the native sequence. This approach shows that it is possible to generate molecules with increased potency and improved plasma stability without major changes of the peptidic nature or the introduction of large conjugates...
December 14, 2017: European Journal of Medicinal Chemistry
Logan C Macdonald, Robin Y Kim, Harley T Kurata, David Fedida
Repolarization of the cardiac action potential is primarily mediated by two voltage-dependent potassium currents: I Kr and I Ks . The voltage-gated potassium channel that gives rise to I Kr, Kv11.1 (hERG), is uniquely susceptible to high-affinity block by a wide range of drug classes. Pore residues Tyr652 and Phe656 are critical to potent drug interaction with hERG. It is considered that the molecular basis of this broad-spectrum drug block phenomenon occurs through interactions specific to the aromatic nature of the side chains at Tyr652 and Phe656...
January 10, 2018: Scientific Reports
Wanting Zeng, Moldir Nukeyeva, Qiumei Wang, Chao Jiang
Pd-Catalyzed o-C-H functionalization of α-phenylglycine, 4-hydroxyphenylglycine and phenylalanine using picolinamide as a directing group is reported. We have developed different protocols for the arylation, alkylation, alkynylation, halogenation, alkoxylation, and acyloxylation of these amino acids. The reactions exhibit high selectivity, broad substrate scope, and compatibility with different functional groups in moderate to high yields. They provide a rapid and efficient access to a variety of phenyl based amino acid derivatives which can be further modified and have broad spectrum of applications in medicinal chemistry...
January 3, 2018: Organic & Biomolecular Chemistry
Anjali Jha, Mothukuri Ganesh Kumar, Hosahudya N Gopi, Kishore M Paknikar
Designing peptide based drugs to target the β-sheet rich toxic intermediates during the aggregation of amyloid-β 1-42 (Aβ1-42) has been a major challenge. In general, β-sheet breaker peptides (BSBPs) are designed to complement the enthalpic interactions with the aggregating protein and entropic effects are usually ignored. Here, we have developed a conformationally constrained cyclic BSBP by the use of an unnatural amino acid and a disulfide bond. We show that our peptide strongly inhibits the aggregation of Aβ1-42 in a concentration-dependent manner...
December 28, 2017: Langmuir: the ACS Journal of Surfaces and Colloids
Subhash Chand, Sriparna Ray, Eranda Wanigasekara, Poonam Yadav, Joshua A Crawford, Daniel W Armstrong, Krishnan Rajeshwar, Brad S Pierce
This study showcases the potential of unnatural amino acids to enable non-natural functions when incorporated in the protein scaffold of heme metalloproteins. For this purpose, a genetically-engineered myoglobin (Mb) mutant was created by incorporating redox-active 3-amino-l-tyrosine (NH2Tyr) into its active site, replacing the distal histidine (H64) with NH2Tyr. In peroxide-shunt assays, this variant exhibits an increased rate of turnover for thioanisole and benzaldehyde oxidation as compared to the wild-type (WT) Mb...
December 22, 2017: Archives of Biochemistry and Biophysics
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