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Current Protocols in Chemical Biology

Alexandra Van Hall-Beauvais, Yi Zhao, Daniel A Urul, Marcus J C Long, Yimon Aye
T-REX (targetable reactive electrophiles and oxidants) enables electrophile targeting in living systems with high spatiotemporal precision and at single-protein-target resolution. T-REX allows functional consequences of individual electrophile signaling events to be directly linked to on-target modifications. T-REX is accomplished by expressing a HaloTagged protein of interest (POI) and introducing a Halo-targetable bioinert photocaged precursor to a reactive electrophilic signal (RES). Light exposure releases the unfettered RES on demand, enabling precision modification of the POI due to proximity...
August 7, 2018: Current Protocols in Chemical Biology
Peter K Foote, Alexander V Statsyuk
PARKIN is a RING-Between-RING (RBR) E3 ligase, which ubiquitinates mitochondrial proteins in response to mitochondrial damage. Ser65 of PARKIN is phosphorylated by kinase PINK1 (pPARKIN), which causes partial PARKIN activation. PINK1 also phosphorylates Ser65 of ubiquitin (pUb), which further activates pPARKIN. Due to the lack of precise and quantitative assays to quantify the activity of PARKIN, there were many conflicting reports on the role of pUb as a PARKIN activator, whether S65E PARKIN is a true phosphomimetic of pPARKIN, and the effect of substrate of PARKIN turnover was also not known...
July 31, 2018: Current Protocols in Chemical Biology
Kiall F Suazo, Alexander K Hurben, Kevin Liu, Feng Xu, Pa Thao, Ch Sudheer, Ling Li, Mark D Distefano
Protein prenylation involves the attachment of a farnesyl or geranylgeranyl group onto a cysteine residue located near the C-terminus of a protein, recognized via a specific prenylation motif, and results in the formation of a thioether bond. To identify putative prenylated proteins and investigate changes in their levels of expression, metabolic labeling and subsequent bioorthogonal labeling has become one of the methods of choice. In that strategy, synthetic analogues of biosynthetic precursors for post-translational modification bearing bioorthogonal functionality are added to the growth medium from which they enter cells and become incorporated into proteins...
July 30, 2018: Current Protocols in Chemical Biology
Christopher Cozens, Vitor B Pinheiro
The B-family polymerases of hyperthermophilic archaea have proven an exceptional platform for engineering polymerases with extended substrate spectra, despite multiple mechanisms for detecting and avoiding incorporation of non-cognate substrates. These polymerases can efficiently synthesize and reverse-transcribe a number of xenonucleic acids (XNAs) that differ significantly from the canonical B-form of DNA. We present here a protocol for hexitol nucleic acid (HNA) synthesis by an engineered Thermococcus gorgonarius polymerase variant, including adaptation for large-scale synthesis and purification, and for other XNAs...
July 24, 2018: Current Protocols in Chemical Biology
Anton Iliuk
Liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) has enabled researchers to analyze complex biological samples with unprecedented depth. It facilitates the identification and quantification of modifications within thousands of proteins in a single large-scale proteomic experiment. Analysis of phosphorylation, one of the most common and important post-translational modifications, has particularly benefited from such progress in the field. Here, detailed protocols are provided for a few well-regarded, common sample preparation methods for an effective phosphoproteomic experiment...
May 31, 2018: Current Protocols in Chemical Biology
Alexander I Taylor, Philipp Holliger
This unit describes the application of "synthetic genetics," i.e., the replication of xeno nucleic acids (XNAs), artificial analogs of DNA and RNA bearing alternative backbone or sugar congeners, to the directed evolution of synthetic oligonucleotide ligands (XNA aptamers) specific for target proteins or nucleic acid motifs, using a cross-chemistry selective exponential enrichment (X-SELEX) approach. Protocols are described for synthesis of diverse-sequence XNA repertoires (typically 1014 molecules) using DNA templates, isolation and panning for functional XNA sequences using targets immobilized on solid phase or gel shift induced by target binding in solution, and XNA reverse transcription to allow cDNA amplification or sequencing...
June 2018: Current Protocols in Chemical Biology
Mia L Huang, Ember M Tota, Stephen Verespy, Kamil Godula
This article describes a protocol for remodeling cells with synthetic glycoprotein and glycolipid mimetics that are functionalized with lipid anchors, allowing for cell surface display of specific glycan structures in predefined nanoscale arrangements. The complex chemical heterogeneity of glycans found on the cell surface or the glycocalyx renders analysis of the individual contributions of glycans difficult. This technique allows for the precise study of individual glycans at different regions of the glycocalyx, and may be useful for interrogating glycan interactions in infection or immunity or in stem cell differentiation...
June 2018: Current Protocols in Chemical Biology
Joshua M Lubner, Jeremy L Balsbaugh, George M Church, Michael F Chou, Daniel Schwartz
Characterizing protein kinase substrate specificity motifs represents a powerful step in elucidating kinase-signaling cascades. The protocol described here uses a bacterial system to evaluate kinase specificity motifs in vivo, without the need for radioactive ATP. The human kinase of interest is cloned into a heterologous bacterial expression vector and allowed to phosphorylate E. coli proteins in vivo, consistent with its endogenous substrate preferences. The cells are lysed, and the bacterial proteins are digested into peptides and phosphoenriched using bulk TiO2 ...
June 2018: Current Protocols in Chemical Biology
Leticia L Torres, Vitor B Pinheiro
Phi29 DNA polymerase (DNAP) is the replicative enzyme of the Bacillus subtilis bacteriophage Phi29. Its extraordinary processivity and its ability to perform isothermal amplification of DNA are central to many molecular biology applications, including high-sensitivity detection and large-scale production of DNA. We present here Phi29 DNAP as an efficient catalyst for the production of various artificial nucleic acids (XNAs) carrying backbone modifications such as 1,5-anhydrohexitol nucleic acid (HNA), 2'-deoxy-2'-fluoro-arabinonucleic acid (FANA), and 2'-fluoro-2'-deoxyribonucleic acid (2'-fluoro-DNA)...
June 2018: Current Protocols in Chemical Biology
Kevin Gorman, Jennifer McGinnis, Brian Kay
Antibodies are useful tools for detecting individual proteins in complex samples and for learning about their location, amount, binding partners, and function in cells. Unfortunately, generating antibodies is time consuming and laborious, and their affinity and/or specificity is often limited. This protocol offers a fast and inexpensive alternative to generate antibody surrogates through phage display of a library of fibronectin type III (FN3) monobody variants and affinity selection for binders. © 2018 by John Wiley & Sons, Inc...
June 2018: Current Protocols in Chemical Biology
Darlene A Pena, Denise M V Pacheco, Paulo S L Oliveira, Maria J M Alves, Deborah Schechtman
The protein kinase C (PKC) family of serine/ threonine kinases has been shown to play active roles as either suppressors or promoters of carcinogenesis in different types of tumors. Using antibodies that preferentially recognize the active conformation of classical PKCs (cPKCs), we have previously shown that in breast cancer samples the expression levels of cPKCs were similar in estrogen receptor-positive (ER+ ) as compared to triple-negative tumors; however, the levels of active cPKCs were different. Determining the activation status of PKCs and other kinases in tumors may thus aid therapeutic decisions...
June 2018: Current Protocols in Chemical Biology
Morgan R Baltz, Erin A Stephens, Matthew P DeLisa
A number of techniques now exist for decreasing the expression of cellular proteins without the need for genomic modification. One such technique involves engineered protein chimeras that combine the ubiquitination activity of E3 ubiquitin ligases with the binding affinity and substrate specificity of designer binding proteins (DBPs). These chimeras, called "ubiquibodies," are capable of selectively and controllably steering virtually any protein to the ubiquitin proteasome pathway (UPP) for degradation, making ubiquibodies a powerful addition to the protein knockout toolbox...
March 2018: Current Protocols in Chemical Biology
Moritz Welter, Andreas Marx
In this unit the preparation and application of enzyme-nucleotide conjugates is depicted. First, a modified nucleoside triphosphate is synthesized bearing a long and flexible linker equipped with a thiol group. The nucleotide is then reacted with maleimide-activated horseradish peroxidase to yield an enzyme-nucleotide conjugate, which due to the long linker, can be used as a substrate by DNA polymerases in primer extension reactions. Finally, an assay based on these findings is described that provides a fast and easy nucleic acid detection and genotyping platform...
March 2018: Current Protocols in Chemical Biology
Zhanar Abil, Andrew D Ellington
Compartmentalized self-replication (CSR) is an emulsion PCR-based method for the selection of DNA polymerases. E. coli host cells expressing a library of DNA polymerases are emulsified so that no more than a single cell is present in a single emulsion droplet. In a subsequent emulsion PCR step, the DNA polymerase protein, as well as the plasmid encoding it are released into the emulsion droplet and the genes that created the most active or abundant polymerase variants are exponentially amplified and can be passed to the next round of CSR...
March 2018: Current Protocols in Chemical Biology
Elena Eremeeva, Piet Herdewijn
An efficient PCR amplification of various templates (short 57-mer, random 67- and 82-mer, and long DNA) with base-modified nucleoside triphosphates is presented here. Using 5-substituted pyrimidine and 7-substituted-7-deaza- or 8-substituted purine nucleoside triphosphates as substrates for thermostable DNA polymerases [Taq and Vent (exo- )], successful PCR amplification of partially or entirely modified DNA libraries and long DNA constructs (up to 1.5 kb) is achieved. Visualization of double-stranded PCR product formation is improved through the use of primers with different fluorescent labels...
March 2018: Current Protocols in Chemical Biology
Jayme L Dahlin, Matthew Cuellar, Gurpreet Singh, Kathryn M Nelson, Jessica Strasser, Todd Rappe, Youlin Xia, Gianluigi Veglia, Michael A Walters
Nonspecific target engagement by test compounds and purported chemical probes is a significant source of assay interference and promiscuous bioactivity in high-throughput screening (HTS) and chemical biology. Most counter-screens for thiol-reactive compounds utilize mass spectrometry or fluorescence detection, and non-proteinaceous reporters like glutathione that may not always approximate the reactivity of protein side-chains. By contrast, a La assay to detect reactive molecules by nuclear magnetic resonance (ALARM NMR) is an industry-developed protein-based [1 H-13 C]-heteronuclear multiple quantum coherence (HMQC) NMR counter-screen to identify nonspecific protein interactions by test compounds by reporting their tendencies to modulate the human La antigen conformation...
March 2018: Current Protocols in Chemical Biology
Michiko Kimoto, Ken-Ichiro Matsunaga, Ichiro Hirao
A novel technology, genetic alphabet expansion, has rapidly advanced through the successful creation of unnatural base pairs that function as a third base pair in replication. Recently, genetic alphabet expansion has been applied to some practical areas. Among them, the application to DNA aptamer generation is a good example of the broad utility of this technology. A hydrophobic unnatural base pair, Ds-Px, which exhibits high fidelity in replication as a third base pair, has been applied to an evolutionary engineering method called SELEX (Systematic Evolution of Ligands by EXponential enrichment) to generate DNA aptamers that bind to targets...
December 14, 2017: Current Protocols in Chemical Biology
Annamaria Ruscito, Erin M McConnell, Anna Koudrina, Ranganathan Velu, Christopher Mattice, Vernon Hunt, Maureen McKeague, Maria C DeRosa
Aptamers, synthetic oligonucleotide-based molecular recognition probes, have found use in a wide array of biosensing technologies based on their tight and highly selective binding to a variety of molecular targets. However, the inherent challenges associated with the selection and characterization of aptamers for small molecule targets have resulted in their underrepresentation, despite the need for small molecule detection in fields such as medicine, the environment, and agriculture. This protocol describes the steps in the selection, sequencing, affinity characterization, and truncation of DNA aptamers that are specific for small molecule targets...
December 14, 2017: Current Protocols in Chemical Biology
Robert W Lee, Mark Mitchnick
When a drug candidate-i.e., a new chemical entity (NCE) or new molecular entity (NME)-is discovered, there is a requirement to identify a vehicle for in vitro and/or in vivo evaluation to assess the activity and/or toxicity of the compound (here we refer to the biologically active compound as the active pharmaceutical ingredient: API). Ideally, this vehicle will not impart any biological activity or any toxicity that would mask or confound the effects of the API. At this early stage in development, and given the high attrition rates of drug candidates in discovery, it does not make sense to fully characterize the API-speed and cost are generally the driving factors...
December 14, 2017: Current Protocols in Chemical Biology
Honghong Wu, Israel Santana, Joshua Dansie, Juan P Giraldo
Plant nanobiotechnology is an interdisciplinary field at the interface of nanotechnology and plant biology that aims to utilize nanomaterials as tools to study, augment or impart novel plant functions. The delivery of nanoparticles to plants in vivo is a key initial step to investigate plant nanoparticle interactions and the impact of nanoparticles on plant function. Quantum dots are smaller than plant cell wall pores, have versatile surface chemistry, bright fluorescence and do not photobleach, making them ideal for the study of nanoparticle uptake, transport, and distribution in plants by widely available confocal microscopy tools...
December 14, 2017: Current Protocols in Chemical Biology
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