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Current Protocols in Chemical Biology

Zilei Liu, Genyi Meng, Taijie Guo, Jiajia Dong, Peng Wu
Sulfur (VI) fluoride exchange (SuFEx) is a new family of click chemistry reactions that relies on readily available sulfuryl fluoride (SO2 F2 ) and ethenesulfonyl fluoride to build diverse chemical structures bearing the SVI -F motif, such as fluorosulfate (-OSO2 F) and sulfonyl fluoride (-SO2 F). These motifs could be useful functional groups and connective linkers in organic synthesis. This unit describes two protocols for performing SuFEx. The first protocol describes an in situ method for rapid generation of arylfluorosulfates in 96-well plates for high-throughput screening...
February 28, 2019: Current Protocols in Chemical Biology
Kenneth H Hu, Marc A Bruce, Jianwei Liu, Manish J Butte
This manuscript details methods to ligate cell-surface receptors on live cells with precise spatiotemporal control using an atomic force microscope (AFM) to deliver ligands. This approach can be used to image cellular responses upon activating T cell receptors when the AFM is mounted on an optical microscope. Moreover, the AFM measures forces generated by the cell during the contact. Using AFM to trigger cellular responses adds an important capability to the field of mechanobiology. We describe how to incorporate anti-CD3 antibodies or other molecules onto an AFM cantilever and how to use AFM to activate T cells...
February 1, 2019: Current Protocols in Chemical Biology
Donaat Kestemont, Piet Herdewijn, Marleen Renders
T4 DNA ligase in high concentrations of certain crowding agents and cosolutes catalyzes the synthesis of a series of backbone-modified oligonucleotides that are difficult to obtain chemically. Backbone-modified nucleic acids are often enzymatically and chemically more stable, making them interesting as potential diagnostic or therapeutic agents, as a biosafety tool, or in nanotechnology. In this article, we describe a small-scale experiment to probe the efficiency of the ligation reaction of modified oligonucleotides in the presence of 3 M betaine and 10% PEG 8000, followed by large-scale ligation with subsequent isolation of the ligated oligonucleotide...
January 28, 2019: Current Protocols in Chemical Biology
Philip A Cistrone, Michael J Bird, Dillon T Flood, Anthony P Silvestri, Jordi C J Hintzen, Darren A Thompson, Philip E Dawson
For over 20 years, native chemical ligation (NCL) has played a pivotal role in enabling total synthesis and semisynthesis of increasingly complex peptide and protein targets. Classical NCL proceeds by chemoselective reaction of two unprotected polypeptide chains in near-neutral-pH, aqueous solution and is made possible by the presence of a thioester moiety on the C-terminus of the N-terminal peptide fragment and a natural cysteine residue on the N-terminus of the C-terminal peptide fragment. The reaction yields an amide bond adjacent to cysteine at the ligation site, furnishing a native protein backbone in a traceless manner...
January 15, 2019: Current Protocols in Chemical Biology
Tomoki Nishioka, Mutsuki Amano, Yasuhiro Funahashi, Daisuke Tsuboi, Yukie Yamahashi, Kozo Kaibuchi
Protein phosphorylation plays a critical role in the regulation of cellular function. Information on protein phosphorylation and the responsible kinases is important for understanding intracellular signaling. A method for in vivo screening of kinase substrates named KIOSS (kinase-oriented substrate screening) has been developed. This protocol provides a method that utilizes phosphoprotein-binding modules such as 14-3-3 protein, the pin1-WW domain, and the chek2-FHA domain as biological filters to successfully enrich phosphorylated proteins related to intracellular signaling rather than housekeeping and/or structural proteins...
January 7, 2019: Current Protocols in Chemical Biology
Lars David Renner
This article describes the design and fabrication of microchambers that are used for the study of bacterial cells. The design allows for the confinement and precise manipulation of bacterial cell shape. The application of fluorescent dyes and fluorescent proteins enables the precise analysis of the localization of biomolecules within confined bacterial cell. This article also outlines three methods to engineer cell shape from a filamentous cell type and from spheroplasts without a cell wall using soft lithography-based technologies...
December 7, 2018: Current Protocols in Chemical Biology
Rachel A Coleman, Darci J Trader
Fluorescence resonance energy transfer (FRET) technology is a useful tool to monitor protein interactions as well as protease activity. We have recently reported a biochemical assay utilizing a FRET reporter peptide to monitor the activity of the 20S catalytic particle (20S CP) of the proteasome. This assay is designed specifically to have increased sensitivity to identify stimulators of the 20S CP, which may hold therapeutic potential to treat protein-accumulation diseases. The protocol described here details the necessary steps in synthesizing the FRET reporter peptide and performing the FRET assay with the 20S CP...
December 2018: Current Protocols in Chemical Biology
Dorsa Parviz, Michael Strano
Due to their two-dimensional structure and unique properties, graphene and its derivatives have been extensively studied for their potential applications in various fields ranging from electronics to composites. Particularly, their high surface area, electrical conductivity, mechanical strength, dispersability in aqueous phase, and possibility of surface modification make them promising candidates for biomedical applications including biosensing, drug delivery, tissue engineering, cell imaging, and therapeutics...
December 2018: Current Protocols in Chemical Biology
Mena Asha Krishnan, Amit Pandit, Venkatesh Chelvam
The development of small molecule ligand-targeted therapeutics is currently of paramount importance for treatment of cancer due to their potential to reduce system toxicity and increase potency of a delivered chemotherapeutic drug. The main aim of a targeted drug-delivery technique is to release the drug cargo selectively into tumor tissues, avoiding off-site toxicity to healthy tissues and organs during chemotherapy. In this strategy, a chemotherapeutic drug is conjugated to a homing ligand, which has high affinity for proteins over-expressed on cancer cells, via a peptide linker and a self-immolative segment that facilitates intracellular release of drug cargo...
December 2018: Current Protocols in Chemical Biology
Mena Asha Krishnan, Sagnik Sengupta, Venkatesh Chelvam
Present treatment strategies focus on minimizing unwanted toxicity to healthy cells during chemotherapeutic treatment. This is achieved by developing strategies to selectively deliver drugs to malignant cells over-expressing specific protein biomarkers. The drugs are attached via a self-immolative linker to a small molecule homing ligand having affinity for protein biomarkers over-expressed during disease states. Several such targeting-ligand drug conjugates have now reached preclinical and clinical trials, and this article aims to show a general methodology to prepare the same...
December 2018: Current Protocols in Chemical Biology
Matthew Fares, Xin Zhang
Proper cellular proteostasis is essential to cellular fitness and viability. Exogenous stress conditions compromise proteostasis and cause aggregation of cellular proteins. We have developed a fluorogenic sensor (AgHalo) to quantify stress-induced proteostasis deficiency. The AgHalo sensor uses a destabilized HaloTag variant to represent aggregation-prone cellular proteins and is equipped with a series of fluorogenic probes that exhibit a fluorescence increase when the sensor forms either soluble oligomers or insoluble aggregates...
November 29, 2018: 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...
September 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...
September 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...
September 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...
September 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...
September 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
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