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Unnatural amino acid

Chuanling Zhang, Jiaqi Lu, Huizhong Su, Jing Yang, Demin Zhou
Cellular protrusion formation capacity is a key feature of developing neurons and many eukaryotic cells. However, the mechanisms underlying membrane growth in protrusion formation are largely unclear. In this study, photo-reactive unnatural amino acid 3-(3-methyl-3H-diazirin-3-yl)-propamino-carbonyl-Nε-l-lysine was incorporated by a genetic code expansion strategy into protrudin, a protein localized in acidic endosomes and in the endoplasmic reticulum, that induces cellular protrusion and neurite formation...
April 21, 2017: Scientific Reports
James S Italia, Yunan Zheng, Rachel E Kelemen, Sarah B Erickson, Partha S Addy, Abhishek Chatterjee
In the last two decades, unnatural amino acid (UAA) mutagenesis has emerged as a powerful new method to probe and engineer protein structure and function. This technology enables precise incorporation of a rapidly expanding repertoire of UAAs into predefined sites of a target protein expressed in living cells. Owing to the small footprint of these genetically encoded UAAs and the large variety of enabling functionalities they offer, this technology has tremendous potential for deciphering the delicate and complex biology of the mammalian cells...
April 15, 2017: Biochemical Society Transactions
László Csaba Bencze, Alina Filip, Gergely Bánóczi, Monica Ioana Toşa, Florin Dan Irimie, Ákos Gellért, László Poppe, Csaba Paizs
This study focuses on the expansion of the substrate scope of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) towards the l-enantiomers of racemic styrylalanines rac-1a-d - which are less studied and synthetically challenging unnatural amino acids - by reshaping the aromatic binding pocket of the active site of PcPAL by point mutations. Ammonia elimination from l-styrylalanine (l-1a) catalyzed by non-mutated PcPAL (wt-PcPAL) took place with a 777-fold lower kcat/KM value than the deamination of the natural substrate, l-Phe...
April 13, 2017: Organic & Biomolecular Chemistry
Song-Min Schinn, William Bradley, Ashtyn Groesbeck, Jeffrey C Wu, Andrew Broadbent, Bradley C Bundy
The incorporation of unnatural amino acids (uAA) can introduce novel functional groups into proteins site-specifically, with important applications in basic sciences and protein engineering. However, uAA incorporation can impact protein expression and functional activity depending on its location within the protein - a process that is not yet completely understood and difficult to predict. Therefore, practical applications often necessitate a time-consuming optimization of uAA location by individual gene cloning, expressions, purification and evaluations for each location tested...
April 11, 2017: Biotechnology and Bioengineering
Yonatan Chemla, Mor Friedman, Mathias Heltberg, Anna Bakhrat, Elad Nagar, Rakefet Schwarz, Mogens Høgh Jensen, Lital Alfonta
The photoautotrophic freshwater cyanobacterium Synechococcus elongatus is widely used as a chassis for biotechnological applications as well as a photosynthetic bacterial model. In this study, a method for expanding the genetic code of this cyanobacterium has been established, thereby allowing the incorporation of unnatural amino acids into proteins. This was achieved through UAG stop codon suppression, using an archaeal pyrrolysyl orthogonal translation system. We demonstrate incorporation of unnatural amino acids into green fluorescent protein with 20 ± 3...
April 12, 2017: Biochemistry
Chen Guo, Heejae Kim, Elisa M Ovadia, Christine M Mourafetis, Mingrui Yang, Wilfred Chen, April M Kloxin
Hydrogels are facile architectures for the controlled presentation of proteins with far-reaching applications, from fundamental biological studies in three-dimensional culture to new regenerative medicine and therapeutic delivery strategies. Here, we demonstrate a versatile approach for spatially-defined presentation of engineered proteins within hydrogels through i) immobilization using bio-orthogonal strain-promoted alkyne-azide click chemistry and ii) dynamic proteasedriven protein release using exogenously applied enzyme...
April 5, 2017: Acta Biomaterialia
Lewis D B Evans, Paul M Bergen, Owain J Bryant, Gillian M Fraser
During assembly of the bacterial flagellum, structural subunits synthesized inside the cell must be exported across the cytoplasmic membrane before they can crystallize into the nascent flagellar structure. This export process is facilitated by a specialized Flagellar Type III Secretion System (fT3SS) located at the base of each flagellum. Here, we describe three methods-isothermal titration calorimetry, photo-crosslinking using unnatural amino acids, and a subunit capture assay-used to investigate the interactions of flagellar structural subunits with the membrane export machinery component FlhB...
2017: Methods in Molecular Biology
Randy P Carney, Yann Thillier, Zsofia Kiss, Amir Sahabi, Jean Carlos Heleno Campos, Alisha Knudson, Ruiwu Liu, David Olivos, Mary Saunders, Lin Tian, Kit S Lam
Membrane active peptides (MAPs) represent a class of short biomolecules that have shown great promise in facilitating intracellular delivery without disrupting cellular plasma membranes. Yet their clinical application has been stalled by numerous factors: off-target delivery, a requirement for high local concentration near cells of interest, degradation en route to the target site, and, in the case of cell-penetrating peptides, eventual entrapment in endolysosomal compartments. The current method of deriving MAPs from naturally occurring proteins has restricted the discovery of new peptides that may overcome these limitations...
April 5, 2017: ACS Combinatorial Science
Michio Sato, Jacob E Dander, Chizuru Sato, Yiu-Sun Hung, Shu-Shan Gao, Man-Cheng Tang, Leibniz Hang, Jaclyn M Winter, Neil K Garg, Kenji Watanabe, Yi Tang
Fungal polyketide synthases (PKSs) can function collaboratively to synthesize natural products of significant structural diversity. Here we present a new mode of collaboration between a highly reducing PKS (HRPKS) and a PKS-nonribosomal peptide synthetase (PKS-NRPS) in the synthesis of oxaleimides from the Penicillium species. The HRPKS is recruited in the synthesis of an olefin-containing free amino acid, which is activated and incorporated by the adenylation domain of the PKS-NRPS. The precisely positioned olefin from the unnatural amino acid is proposed to facilitate a scaffold rearrangement of the PKS-NRPS product to forge the maleimide and succinimide cores of oxaleimides...
April 5, 2017: Journal of the American Chemical Society
Wen-Xue Jiang, Xin-Hua Gu, Xu Dong, Chun Tang
Lanthanoid pseudo-contact shift (PCS) provides long-range structural information between a paramagnetic tag and protein nuclei. However, for proteins with native cysteines, site-specific attachment may only utilize functional groups orthogonal to sulfhydryl chemistry. Here we report two lanthanoid probes, DTTA-C3-yne and DTTA-C4-yne, which can be conjugated to an unnatural amino acid pAzF in the target protein via azide-alkyne cycloaddition. Demonstrated with ubiquitin and cysteine-containing enzyme EIIB, we show that large PCSs of distinct profiles can be generated for each tag/lanthanoid combination...
April 1, 2017: Journal of Biomolecular NMR
Yuan Wang, Jingjing Zhou, Xin Liu, Long Zhao, Zhaojuan Wang, Xianghui Zhang, Kezhou Wang, Linqing Wang, Rui Wang
We previously reported a series of novel endomorphin analogs with unnatural amino acid modifications. These analogs display good binding affinity and functional activity toward the μ opioid receptor (MOP). In the present study, we further investigated the spinal antinociceptive activity of these compounds. The analogs were potent in several nociceptive models. Opioid antagonists and antibodies against several endogenous opioid peptides were used to determine the mechanisms of action of these peptides. Intrathecal pretreatment with naloxone and β-funaltrexamine (β-FNA) effectively inhibited analog-induced analgesia, demonstrating that activity of the analogs is regulated primarily through MOP...
March 29, 2017: Peptides
Chilakapati Madhu, Chandrashekhar Voshavar, K Rajasekhar, Thimmaiah Govindaraju
An unnatural CDP-amino acid (kd) is used in the design and synthesis of novel cell penetrating peptidomimetic (Kkd-5). The balanced cationic nature and structural rigidity of Kkd-5 resulted in serum stability, non-toxicity to cells, and interaction with membrane and DNA, all of which facilitated efficient cellular uptake and DNA delivery.
April 11, 2017: Organic & Biomolecular Chemistry
Mattias Brofelth, Lars Wagner Städe, Anna Isinger Ekstrand, Linn Petersson Edfeldt, Rebeka Kovačič, Thorbjørn Terndrup Nielsen, Kim Lambertsen Larsen, Laurent Duroux, Christer Wingren
Recombinant antibody libraries can provide a source of renewable and high-performing binders tailored for use in affinity proteomics. In this context, the process of generating site-specific 1:1 tagging/functionalization and/or orientated surface immobilization of antibodies has, however, proved to be challenging. Hence, novel ways of generating such engineered antibodies for use in affinity proteomics could have a major impact on array performance. In this study, we have further tailored the design of human recombinant scFv antibodies for site-specific photocoupling through the use of an unnatural amino acid (UAA) and the Dock'n'Flash technology...
March 22, 2017: Biochimica et Biophysica Acta
Wei Niu, Jiantao Guo
Fluorescent proteins of different colors are useful probes to study protein structure and function, and to investigate cellular events and conditions. Large efforts have focused on engineering new properties into fluorescent proteins via rational design and directed evolution. In addition to applications in imaging of protein expression level and subcellular localization, fluorescent proteins have been increasingly engineered to act as biosensors to track concentrations of small-molecule metabolites, enzyme activities, and protein conformational changes in living cells...
2017: Methods in Enzymology
Xiao Lin, Allen Chi Shing Yu, Ting Fung Chan
This year marks the 48th anniversary of Francis Crick's seminal work on the origin of the genetic code, in which he first proposed the "frozen accident" hypothesis to describe evolutionary selection against changes to the genetic code that cause devastating global proteome modification. However, numerous efforts have demonstrated the viability of both natural and artificial genetic code variations. Recent advances in genetic engineering allow the creation of synthetic organisms that incorporate noncanonical, or even unnatural, amino acids into the proteome...
March 14, 2017: Life
Xiuling Li, Christopher G Nelson, Rajesh R Nair, Lori Hazlehurst, Tina Moroni, Pablo Martinez-Acedo, Alex R Nanna, David Hymel, Terrence R Burke, Christoph Rader
Selenomabs are engineered monoclonal antibodies with one or more translationally incorporated selenocysteine residues. The unique reactivity of the selenol group of selenocysteine permits site-specific conjugation of drugs. Compared with other natural and unnatural amino acid and carbohydrate residues that have been used for the generation of site-specific antibody-drug conjugates, selenocysteine is particularly reactive, permitting fast, single-step, and efficient reactions under near physiological conditions...
April 20, 2017: Cell Chemical Biology
Anastasia Loktev, Uwe Haberkorn, Walter Mier
The lack of specificity of traditional cytotoxic drugs triggers the development of anticancer agents with high selectivity to tumor-specific proteins. The unveiling of target structures such as EGFR or Her2 allows the focused development of novel therapies and has strongly advanced tumor treatment. Tumor-specific high-affinity ligands can be identified by using display techniques such as phage, yeast surface, ribosome and mRNA display. These techniques enable the screening of huge libraries, consequently providing a valuable alternative to rational drug development...
March 16, 2017: Current Medicinal Chemistry
Brad D Maxwell, Van Ly, Barry Brock, Robert Dodge, Mark Tirmenstein, Jacqueline Calvano
PEGylation is a proven approach to prolonging the duration of action and enhancing biophysical solubility and stability of peptides. 4-Acetylphenylalanine is a novel amino acid with a ketone side chain that is uniquely reactive in proteins. The ketone functionality can react with an amino-oxy functionalized PEG polymer to form a stable oxime adduct of the protein. One concern with using unnatural amino acids, such as 4-acetyl-phenylalanine, is the possibility of it being cleaved from the peptide and becoming incorporated into endogenous proteins...
March 8, 2017: Journal of Labelled Compounds & Radiopharmaceuticals
Zhao Yang, Fan Yang, Daolai Zhang, Zhixin Liu, Amy Lin, Chuan Liu, Peng Xiao, Xiao Yu, Jin-Peng Sun
Seven transmembrane G protein-coupled receptors (GPCRs) are often phosphorylated at the C-terminus and on intracellular loops in response to various extracellular stimuli. Phosphorylation of GPCRs by GPCR kinases (GRKs) and certain other kinases can promote the recruitment of arrestin molecules. The arrestins critically regulate GPCR functions, not only by mediating receptor desensitization and internalization, but also by redirecting signalling to G protein-independent pathways via interactions with numerous downstream effector molecules...
February 28, 2017: Molecular Pharmacology
Marcin Poreba, Aleksandra Szalek, Wioletta Rut, Paulina Kasperkiewicz, Izabela Rutkowska-Wlodarczyk, Scott J Snipas, Yoshifumi Itoh, Dusan Turk, Boris Turk, Christopher M Overall, Leszek Kaczmarek, Guy S Salvesen, Marcin Drag
Internally quenched fluorescent (IQF) peptide substrates originating from FRET (Förster Resonance Energy Transfer) are powerful tool for examining the activity and specificity of proteases, and a variety of donor/acceptor pairs are extensively used to design individual substrates and combinatorial libraries. We developed a highly sensitive and adaptable donor/acceptor pair that can be used to investigate the substrate specificity of cysteine proteases, serine proteases and metalloproteinases. This novel pair comprises 7-amino-4-carbamoylmethylcoumarin (ACC) as the fluorophore and 2,4-dinitrophenyl-lysine (Lys(DNP)) as the quencher...
February 23, 2017: Scientific Reports
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