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Unnatural amino acid

Xin Shang, Rui Lai, Xi Song, Hui Li, Wei Niu, Jiantao Guo
The 1,3-dipolar cycloaddition reaction between an alkene and a tetrazole represents one elegant and rare example of fluorophore-forming bioorthogonal chemistry. This is an attractive reaction for imaging applications in live cells that requires less intensive washing steps and/or needs spatiotemporal resolutions. In the present work, as an effort to improve the fluorogenic property of the alkene-tetrazole reaction, an aromatic alkene (styrene) was investigated as the dipolarophile. Over 30-fold improvement in quantum yield of the reaction product was achieved in aqueous solution...
October 12, 2017: Bioconjugate Chemistry
Nicolas Panel, Young Joo Sun, Ernesto J Fuentes, Thomas Simonson
PDZ domains generally bind short amino acid sequences at the C-terminus of target proteins, and short peptides can be used as inhibitors or model ligands. Here, we used experimental binding assays and molecular dynamics simulations to characterize 51 complexes involving the Tiam1 PDZ domain and to test the performance of a semi-empirical free energy function. The free energy function combined a Poisson-Boltzmann (PB) continuum electrostatic term, a van der Waals interaction energy, and a surface area term. Each term was empirically weighted, giving a Linear Interaction Energy or "PB/LIE" free energy...
2017: Frontiers in Molecular Biosciences
Wooseok Ko, Sanggil Kim, Hyun Soo Lee
Periplasmic binding proteins (PBPs) are members of a widely distributed protein superfamily found in bacteria and archaea, and are involved in the cellular uptake of solutes. In this report, a leucine-binding PBP was engineered to detect l-Leu based on a fluorescence resonance energy transfer (FRET) change upon ligand binding. A fluorescent unnatural amino acid, l-(7-hydroxycoumarin-4-yl)ethylglycine (CouA), was genetically incorporated into the protein as a FRET donor, and a yellow fluorescent protein (YFP) was fused with its N-terminus as a FRET acceptor...
October 10, 2017: Organic & Biomolecular Chemistry
Isabel Betancor-Fernández, David J Timson, Eduardo Salido, Angel L Pey
Mutations causing single amino acid exchanges can dramatically affect protein stability and function, leading to disease. In this chapter, we will focus on several representative cases in which such mutations affect protein stability and function leading to cancer. Mutations in BRAF and p53 have been extensively characterized as paradigms of loss-of-function/gain-of-function mechanisms found in a remarkably large fraction of tumours. Loss of RB1 is strongly associated with cancer progression, although the molecular mechanisms by which missense mutations affect protein function and stability are not well known...
October 10, 2017: Handbook of Experimental Pharmacology
Meilin Tian, Shixin Ye
Genetic code expansion exploiting unnatural amino acids (Uaas) is a powerful technique to create novel protein function in vivo. Here, we provide a protocol for the incorporation of two UV-sensitive crosslinking Uaas into NMDA receptors (NMDARs), a type of glutamate-gated ion channels mediating fast synaptic transmission. Through heterologous expression in Xenopus laevis oocytes, we have identified light-sensitive NMDARs of GluN2B subtype by using the two-electrode voltage electrophysiology measurement in combination with online-UV application...
2017: Methods in Molecular Biology
Lei Wang
Expansion of the genetic code allows unnatural amino acids (Uaas) to be site-specifically incorporated into proteins in live biological systems, thus enabling novel properties selectively introduced into target proteins in vivo for basic biological studies and for engineering of novel biological functions. Orthogonal components including tRNA and aminoacyl-tRNA synthetase (aaRS) are expressed in live cells to decode a unique codon (often the amber stop codon UAG) as the desired Uaa. Initially developed in E...
October 6, 2017: Accounts of Chemical Research
Nanxi Wang, Zhe Yuan, Wei Niu, Qingsheng Li, Jiantao Guo
While the combined antiretroviral therapy has resulted in a significant decrease in HIV-1 related morbidity and mortality, the HIV-1 pandemic has not been substantially averted. To curtail the 2.4 million new infections each year, a prophylactic HIV-1 vaccine is urgently needed. This review first summarizes four major completed clinical efficacy trials of prophylactic HIV-1 vaccine and their outcomes. Next, it discusses several other approaches that have not yet advanced to clinical efficacy trials, but provided valuable insights into vaccine design...
March 2017: Journal of Chemical Technology and Biotechnology
Marko Cigler, Thorsten G Müller, Daniel Horn-Ghetko, Marie-Kristin von Wrisberg, Maximilian Fottner, Roger S Goody, Aymelt Itzen, Matthias P Müller, Kathrin Lang
The characterization of low-affinity protein complexes is challenging due to their dynamic nature. Here we present a method to stabilize transient protein complexes in vivo by generating a covalent and conformationally flexible bridge between the interaction partners. A highly active pyrrolysyl tRNA synthetase mutant directs the incorporation of unnatural amino acids bearing bromoalkyl moieties (BrCnK) into proteins. We demonstrate for the first time that low-affinity protein complexes between BrCnK-containing proteins and their binding partners can be stabilized in vivo in bacterial and mammalian cells...
September 28, 2017: Angewandte Chemie
Bauke Albada, Nils Metzler-Nolte
Resistance of pathogenic bacteria against currently marketed antibiotics is again increasing. To meet the societal need for effective cures, scientists are faced with the challenge of developing more potent but equally bacteria-specific drugs. Currently, most efforts are directed toward the modification of existing antibiotics, but ideally, compounds with a new mode of action are required. In this Account, we detail our findings in the area of novel metal-based antibiotics. Our strategy is based on the modification of simple antimicrobial peptides (AMPs) with organometallic agents, resulting in organometallic AMPs (OM-AMPs)...
October 17, 2017: Accounts of Chemical Research
John J Ferrie, Naoya Ieda, Conor M Haney, Christopher R Walters, Itthipol Sungwienwong, Jimin Yoon, E James Petersson
Site-specific fluorescence probes can be used to measure distances within proteins when used as part of a Förster resonance energy transfer (FRET) pair. Here we report the synthesis of a coumarin maleimide (Mcm-Mal) that is fluorogenic upon reaction with cysteine. We demonstrate that cysteine, acridonylalanine (Acd) double mutant proteins can be produced by unnatural amino acid mutagenesis and reacted with Mcm-Mal to generate Mcm/Acd labeled proteins for FRET studies. The Mcm/Acd FRET pair is minimally-perturbing, easy to install, and well-suited to studying protein distances in the 15-40 Å range...
October 5, 2017: Chemical Communications: Chem Comm
Xin X Zhou, Xinzhi Zou, Hokyung K Chung, Yuchen Gao, Yanxia Liu, Lei S Qi, Michael Z Lin
Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination...
September 29, 2017: ACS Chemical Biology
Jian Tang, Yadong He, Hongfei Chen, Wangjian Sheng, Huan Wang
Cyclic peptides have attracted increasing attention in recent years due to their ability to inhibit protein-protein interactions. Current strategies to prepare cyclic peptides often rely on functional amino acid side chains or the incorporation of unnatural amino acids, thus limiting their structural diversity. Here, we describe the development of a highly versatile peptide macrocyclization strategy through a palladium-catalyzed C(sp(3))-H activation and the synthesis of cyclic peptides featuring unique hydrocarbon linkages between the β-carbon of amino acids and the aromatic side chains of Phe and Trp...
June 1, 2017: Chemical Science
Satoru Horiya, Jennifer K Bailey, Isaac J Krauss
Directed evolution is a useful method for the discovery of nucleic acids, peptides, or proteins that have desired binding abilities or functions. Because of the abundance and importance of glycosylation in nature, directed evolution of glycopeptides and glycoproteins is also highly desirable. However, common directed evolution platforms such as phage-, yeast-, or mammalian-cell display are limited for these applications by several factors. Glycan structure at each glycosylation site is not genetically encoded, and yeast and mammalian cells produce a heterogeneous mixture of glycoforms at each site on the protein...
2017: Methods in Enzymology
Yi Yang, Haiping Song, Dan He, Shuai Zhang, Shizhong Dai, Xiao Xie, Shixian Lin, Ziyang Hao, Huangtao Zheng, Peng R Chen
Although protein-protein interactions (PPIs) have crucial roles in virtually all cellular processes, the identification of more transient interactions in their biological context remains challenging. Conventional photo-cross-linking strategies can be used to identify transient interactions, but these approaches often suffer from high background due to the cross-linked bait proteins. To solve the problem, we have developed membrane-permeable releasable photo-cross-linkers that allow for prey-bait separation after protein complex isolation and can be installed in proteins of interest (POIs) as unnatural amino acids...
October 2017: Nature Protocols
Marcin Poreba, Guy S Salvesen, Marcin Drag
Many biologically and chemically based approaches have been developed to design highly active and selective protease substrates and probes. It is, however, difficult to find substrate sequences that are truly selective for any given protease, as different proteases can demonstrate a great deal of overlap in substrate specificities. In some cases, better enzyme selectivity can be achieved using peptide libraries containing unnatural amino acids such as the hybrid combinatorial substrate library (HyCoSuL), which uses both natural and unnatural amino acids...
October 2017: Nature Protocols
Hafsteinn Rannversson, Jacob Andersen, Benny Bang-Andersen, Kristian Strømgaard
In spite of the important role of the human serotonin transporter (hSERT) in depression treatment, the molecular details of how antidepressant drugs bind are still not completely understood, in particular those related to potential high- and low-affinity binding sites in hSERT. Here, we utilize amber codon suppression in hSERT to encode the photocrosslinking unnatural amino acid p-azido-L-phenylalanine into the suggested high- and low-affinity binding sites. We then employ UV-induced crosslinking with azF to map the binding site of escitalopram and paroxetine, two prototypical selective serotonin reuptake inhibitors (SSRIs)...
September 14, 2017: ACS Chemical Biology
Zahra B Dizicheh, Nicholas Halloran, William Asma, Giovanna Ghirlanda
Iron-sulfur proteins are one of the most abundant and functionally pliable redox proteins found in all living organisms. Because of their crucial role in mediating electron transfer processes, minimalist model systems have been developed as a proxy to study natural Fe-S redox proteins and to dissect rules to enable tuning of their redox and electron transfer activities. This goal has been pursued through computational design, mutagenesis in the first and second coordination sphere, metal substitution, cofactor replacement, and the use of unnatural amino acids to stabilize a given cluster...
2017: Methods in Enzymology
Ryan K Spencer, Allon I Hochbaum
Coiled coils are a robust motif for exploring amino acid interactions, generating unique supramolecular structures, and expanding the functional properties of biological materials. A recently discovered antiparallel coiled-coil hexamer (ACC-Hex, peptide 1) exhibits a unique interaction in which Phe and Ile residues from adjacent α-helices interact to form a Phe-Ile zipper within the hydrophobic core. Analysis of the X-ray crystallographic structure of ACC-Hex suggests that the stability of the six-helix bundle relies on specific interactions between the Phe and Ile residues...
September 25, 2017: Biochemistry
Bo Qian, Shaowei Chen, Ting Wang, Xinhao Zhang, Hongli Bao
Intermolecular carboamination of olefins with general alkyl groups is an unsolved problem. Diastereoselective carboamination of acyclic olefins represents an additional challenge in intermolecular carboaminations. We have developed a general alkylamination of vinylarenes and the unprecedented diastereoselective anti-carboamination of unsaturated esters, generating amines and unnatural β-amino acids. This alkylamination is enabled by difunctional alkylating reagents and the iron catalyst. Alkyl diacyl peroxides, readily synthesized from aliphatic acids, serve as both alkylating reagents and internal oxidizing agents...
September 8, 2017: Journal of the American Chemical Society
Norman Metanis, Reem Mousa, Rebecca Notis Dardashti
Selenocysteine, the selenium-containing analog of cysteine, is the twenty-first proteinogenic amino acid. Since its discovery almost fifty years ago, it has been exploited in unnatural systems more often than in natural systems. Selenocysteine chemistry has attracted the attention of many chemists in the field of chemical biology due to its high reactivity and resulting utility in various applications including chemical modification, chemical protein (semi-)synthesis, and protein folding, to name a few. In this review, we will focus on the chemistry of selenium and selenocysteine and their utility in protein chemistry...
August 30, 2017: Angewandte Chemie
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