pinecone/core-2020-05-10-deduplication huggingface dataset:
Kinetic analysis of the interaction of Mos1 transposase with its inverted terminal repeats reveals new insight into the protein-DNA complex assembly.

International audienceTransposases are specific DNA-binding proteins that promote the mobility of discrete DNA segments. We used a combination of physicochemical approaches to describe the association of MOS1 (an eukaryotic transposase) with its specific target DNA, an event corresponding to the first steps of the transposition cycle. Because the kinetic constants of the reaction are still unknown, we aimed to determine them by using quartz crystal microbalance on two sources of recombinant MOS1: one produced in insect cells and the other produced in bacteria. The prokaryotic-expressed MOS1 showed no cooperativity and displayed a Kd of about 300 nM. In contrast, the eukaryotic-expressed MOS1 generated a cooperative system, with a lower Kd (∼ 2 nm). The origins of these differences were investigated by IR spectroscopy and AFM imaging. Both support the conclusion that prokaryotic- and eukaryotic-expressed MOS1 are not similarly folded, thereby resulting in differences in the early steps of transposition
Next-generation sequencing studies guide the design of pyrrole-imidazole polyamides with improved binding specificity by the addition of β-alanine.

The identification of binding sites for small molecules in genomic DNA is important in various applications. Previously, we demonstrated rapid transcriptional activation by our small molecule SAHA-PIP. However, it was not clear whether the strong biological effects exerted by SAHA-PIP were attributable to its binding specificity. Here, we used high-throughput sequencing (Bind-n-seq) to determine the binding specificity of SAHA-PIPs. Sequence specificity bias was determined for SAHA-PIPs (3 and 4), and this showed enhanced 6 bp sequence-specific binding compared with hairpin PIPs (1 and 2). This finding allowed us to investigate the role of the β-alanine that links SAHA to PIP, and led in turn to the design of ββ-PIPs (5 and 6), which showed enhanced binding specificity. Overall, we demonstrated the importance of β-moieties for the binding specificity of PIPs and the use of cost-effective high-throughput screening of these small molecules for binding to the DNA minor groove
Advancing small-molecule-based chemical biology with next-generation sequencing technologies

Next-generation-sequencing (NGS) technologies enable us to obtain extensive information by deciphering millions of individual DNA sequencing reactions simultaneously. The new DNA-sequencing strategies exceed their precursors in output by many orders of magnitude, resulting in a quantitative increase in valuable sequence information that could be harnessed for qualitative analysis. Sequencing on this scale has facilitated significant advances in diverse disciplines, ranging from the discovery, design, and evaluation of many small molecules and relevant biological mechanisms to maturation of personalized therapies. NGS technologies that have recently become affordable allow us to gain in-depth insight into small-molecule-triggered biological phenomena and empower researchers to develop advanced versions of small molecules. In this review we focus on the overlooked implications of NGS technologies in chemical biology, with a special emphasis on small-molecule development and screening
Directed Divergent Evolution of a Thermostable D-Tagatose Epimerase towards Improved Activity for Two Hexose Substrates

Functional promiscuity of enzymes can often be harnessed as the starting point for the directed evolution of novel biocatalysts. Here we describe the divergent morphing of an engineered thermostable variant (Var8) of a promiscuous D-tagatose epimerase (DTE) into two efficient catalysts for the C3 epimerization of D-fructose to D-psicose and of L-sorbose to L-tagatose. Iterative single-site randomization and screening of 48 residues in the first and second shells around the substrate-binding site of Var8 yielded the eight-site mutant IDF8 (ninefold improved kcat for the epimerization of D-fructose) and the six-site mutant ILS6 (14-fold improved epimerization of L-sorbose), compared to Var8. Structure analysis of IDF8 revealed a charged patch at the entrance of its active site; this presumably facilitates entry of the polar substrate. The improvement in catalytic activity of variant ILS6 is thought to relate to subtle changes in the hydration of the bound substrate. The structures can now be used to select additional sites for further directed evolution of the ketohexose epimerase
Rapid Parallel Synthesis of Bioactive Folded Cyclotides by Using a Tea‐Bag Approach

We report here the first rapid parallel production of bioactive folded cyclotides by using Fmoc‐based solid‐phase peptide synthesis in combination with a “tea‐bag” approach. Using this approach, we efficiently synthesized 15 analogues of the CXCR4 antagonist cyclotide MCo‐CVX‐5c. Cyclotides were synthesized in a single‐pot, cyclization/folding reaction in the presence of reduced glutathione. Natively folded cyclotides were quickly purified from the cyclization/folding crude mixture by activated thiol Sepharose‐based chromatography. The different folded cyclotide analogues were then tested for their ability to inhibit the CXCR4 receptor in a cell‐based assay. The results indicated that this approach can be used for the efficient chemical synthesis of libraries of cyclotides with improved biological properties that can be easily interfaced with solution or cell‐based assays for rapid screening.Let the cyclotides flood out: Bioactive folded MCoTI‐based cyclotides can be efficiently produced in parallel by using a “tea‐bag” approach in combination with highly efficient cyclization/folding protocols. This approach was successfully used to produce a small library of bioactive cyclotides with CXCR4 antagonistic activity
Formation and Abundance of 5-Hydroxymethylcytosine in RNA

RNA methylation is emerging as a regulatory RNA modification that could have important roles in the control and coordination of gene transcription and protein translation. Herein, we describe an in vivo isotope-tracing methodology to demonstrate that the ribonucleoside 5-methylcytidine (m5C) is subject to oxidative processing in mammals, forming 5-hydroxymethylcytidine (hm5C) and 5-formylcytidine (f5C). Furthermore, we have identified hm5C in total RNA from all three domains of life and in polyA-enriched RNA fractions from mammalian cells. This suggests m5C oxidation is a conserved process that could have critical regulatory functions inside cells.This work was supported by the Cambridge PhD Training Programme
in Chemical Biology and Molecular Medicine and the
Wellcome Trust (grant number 099232/Z/12/Z). Dr. Donna Bond
is thanked for the provision of A. thaliana total RNA and Drs.
Santiago Uribe-Lewis and Adele Murrell are acknowledged for
the labelled mouse tissue supply.This is the final published version. It first appeared at http://onlinelibrary.wiley.com/doi/10.1002/cbic.201500013/abstract;jsessionid=61B9B3D8937FE50CFA4954A4C4B445B6.f02t04
Orthogonal Translation Meets Electron Transfer: In Vivo Labeling of Cytochrome c for Probing Local Electric Fields

Cytochrome c (cyt c), a redox protein involved in diverse fundamental biological processes, is among the most traditional model proteins for analyzing biological electron transfer and protein dynamics both in solution and at membranes. Studying the role of electric fields in energy transduction mediated by cyt c relies upon appropriate reporter groups. Up to now these had to be introduced into cyt c by in vitro chemical modification. Here, we have overcome this restriction by incorporating the noncanonical amino acid p-cyanophenylalanine (pCNF) into cyt c in vivo. UV and CD spectroscopy indicate preservation of the overall protein fold, stability, and heme coordination, whereas a small shift of the redox potential was observed by cyclic voltammetry. The C≡N stretching mode of the incorporated pCNF detected in the IR spectra reveals a surprising difference, which is related to the oxidation state of the heme iron, thus indicating high sensitivity to changes in the electrostatics of cyt c.Fil: Völler, Jan. Technishe Universitat Berlin; AlemaniaFil: Biava, Hernan Daniel. Technishe Universitat Berlin; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Química Rosario; ArgentinaFil: Koksch, Beate. Freie Universitä t Berlin; AlemaniaFil: Hildebrandt, Peter. Technishe Universitat Berlin; AlemaniaFil: Budisa, Nediljko. Technishe Universitat Berlin; Alemani
Preparation of fluorescent microcystin derivatives by direct arginine labelling and their biological evaluation

Microcystin is the most prevalent toxin produced by cyanobacteria and poses a severe threat to livestock, humans and entire ecosystems. We report the preparation of a series of fluorescent microcystin derivatives by direct arginine labelling of the unprotected peptides at the guanidinium side chain. This new method allows a simple late-stage diversification strategy for native peptides devoid of protecting groups under mild conditions. A series of fluorophores were conjugated to microcystin-LR in good to very good yield. The fluorescent probes displayed biological activity comparable to that of unlabelled microcystin, in both phosphatase inhibition assays and toxicity tests on the crustacean Thamnocephalus platyurus. In addition, we demonstrate that the fluorescent probes penetrated Huh7 cells. Whole-animal imaging was performed on T. platyurus: labelled compound was mainly observed in the digestive tract
Handicap-recover evolution leads to a chemically versatile, nucleophile-permissive protease

Mutation of the TEV protease nucleophile from cysteine to serine causes a ~10e4-fold activity loss. Ten rounds of directed evolution of the mutant, TEVSer, overcame the detrimental nucleophile exchange to recover near-wild-type activity in the mutant TEVSerX. Rather than respecialising TEV to the new nucleophile, all enzymes along the evolutionary trajectory retained the ability to also use the original cysteine nucleophile. Thus the adaptive evolution of TEVSer is paralleled by a neutral trajectory for TEVCys, in which mutations that increase serine nucleophile reactivity hardly affect the reactivity of cysteine. This apparent nucleophile permissiveness explains how nucleophile switches can occur in the phylogeny of the chymotrypsin-like protease PA superfamily. Despite changing the key component of its chemical mechanism, the evolved variants TEVSerX and TEVCysX have similar activities, which could potentially facilitate escape from adaptive conflict to enable active site evolution.We acknowledge financial support from the Biotechnology and Biological Sciences Research Council and MedImmune. F. H. is an ERC Starting Investigator.This is the final version of the article. It first appeared from Wiley at http://dx.doi.org/10.1002/cbic.20150029
Investigating the Role of Conformational Effects on Laccase Stability and Hyperactivation under Stress Conditions

Fungal laccase from Steccherinum ochraceum 1833 displays remarkable stability under different harsh conditions: organic/buffer mixtures, thermal treatment, and microwave radiation. The behavior is particularly significant in the light of the sharp inactivation observed for two different fungal laccases. Laccase from S. ochraceum 1833 also displays hyperactivation under mild thermal treatment (60 °C). Molecular dynamics simulations at 80 °C explained how this laccase retains the geometry of the electron transfer pathway, thereby assuring electron transfer through the copper ions and thus maintaining its catalytic activity at high temperature. Spectroscopic studies revealed that the thermal activation corresponds to specific conformational changes in the protein. The results indicate that this laccase is potentially applicable under denaturing conditions that might be beneficial for the biotransformation of recalcitrant substrates
Streptocollin, a Type IV Lanthipeptide Produced by Streptomyces collinus Tü 365

Lanthipeptides are ribosomally synthesized and post-translationally modified microbial secondary metabolites. Here, we report the identification and isolation of streptocollin from Streptomyces collinus Tü 365, a new member of class IV lanthipeptides. Insertion of the constitutive ermE* promoter upstream of the lanthipeptide synthetase gene stcL resulted in peptide production. The streptocollin gene cluster was heterologously expressed in S. coelicolor M1146 and M1152 with 3.5- and 5.5-fold increased yields, respectively. The structure and ring topology of streptocollin were determined by high resolution MS/MS analysis. Streptocollin contains four macrocyclic rings, with one lanthionine and three methyllanthionine residues. To the best of our knowledge, this is the first report on the isolation of a class IV lanthipeptide in preparative amounts, and on the successful heterologous expression of a class IV lanthipeptide gene cluster
Synthesis of 1‐Naphthol by a Natural Peroxygenase engineered by Directed Evolution

This is the peer reviewed version of the following article, which has been published in final form at 10.1002/cbic.201500493. This article may be used for non-commercial purposes in accordance With Wiley-VCH Terms and Conditions for self-archivingThere is an increasing interest in enzymes that catalyze the hydroxylation of naphthalene under mild conditions and with minimal requirements. To address this challenge, an extracellular fungal aromatic peroxygenase with mono(per)oxygenase activity was engineered to convert naphthalene selectively into 1-naphthol. Mutant libraries constructed by random mutagenesis and DNA recombination were screened for peroxygenase activity on naphthalene together with quenching of the undesired peroxidative activity on 1-naphthol (one-electron oxidation). The resulting double mutant (G241D-R257K) obtained from this process was characterized biochemically and computationally. The conformational changes produced by directed evolution improved the substrate's catalytic position. Powered exclusively by catalytic concentrations of H2O2, this soluble and stable biocatalyst has a total turnover number of 50 000, with high regioselectivity (97 %) and reduced peroxidative activity.We thank Paloma Santos Moriano (ICP, CSIC, Spain) for assistance with the HPLC and LC/MS analysis, and Jesper Vind (Novozymes, Denmark) and Angel T. Martinez (CIB, CSIC, Spain) for helpful discussions. This work was supported by the European Commission projects Indox-FP7-KBBE-2013-7-613549 and Cost-Action CM1303-Systems Biocatalysis, and the National Projects Dewry [BIO201343407-R], Cambios [RTC-2014-1777-3] and OXYdesign [CTQ2013-48287-R].Peer ReviewedPostprint (author's final draft
Derivatisable cyanobactin analogues : a semisynthetic approach

This work was supported by the European Research Council (339367), UK Biotechnology and Biological Sciences Research Council (K015508/1) and the Wellcome Trust (Triple TOF 5600 mass spectrometer (094476), the MALDI TOF-TOF Analyser (079272AIA), 700 NMR and supported G.M. (097831)). J.H.N. is a Royal Society Wolfson Merit Award Holder and 1000 talent scholar at Sichuan University.Many natural cyclic peptides have potent and potentially useful biological activities. Their use as therapeutic starting points is often limited by the quantities available, the lack of known biological targets and the practical limits on diversification to fine-tune their properties. We report the use of enzymes from the cyanobactin family to heterocyclise and macrocyclise chemically synthesised substrates so as to allow larger-scale syntheses and better control over derivatisation. We have made cyclic peptides containing orthogonal reactive groups, azide or dehydroalanine, that allow chemical diversification, including the use of fluorescent labels that can help in target identification. We show that the enzymes are compatible and efficient with such unnatural substrates. The combination of chemical synthesis and enzymatic transformation could help renew interest in investigating natural cyclic peptides with biological activity, as well as their unnatural analogues, as therapeutics.Publisher PDFPeer reviewe
The amyloid fibril-forming properties of the amphibian antimicrobial peptide uperin 3.5

The amphibian skin is a vast resource for bioactive peptides, which form the basis of the animals' innate immune system. Key components of the secretions of the cutaneous glands are antimicrobial peptides (AMPs), which exert their cytotoxic effects often as a result of membrane disruption. It is becoming increasingly evident that there is a link between the mechanism of action of AMPs and amyloidogenic peptides and proteins. In this work, we demonstrate that the broad-spectrum amphibian AMP uperin 3.5, which has a random-coil structure in solution but adopts an α-helical structure in membrane-like environments, forms amyloid fibrils rapidly in solution at neutral pH. These fibrils are cytotoxic to model neuronal cells in a similar fashion to those formed by the proteins implicated in neurodegenerative diseases. The addition of small quantities of 2,2,2-trifluoroethanol accelerates fibril formation by uperin 3.5, and is correlated with a structural stabilisation induced by this co-solvent. Uperin 3.5 fibril formation and the associated cellular toxicity are inhibited by the polyphenol (-)-epigallocatechin-3-gallate (EGCG). Furthermore, EGCG rapidly dissociates fully formed uperin 3.5 fibrils. Ion mobility-mass spectrometry reveals that uperin 3.5 adopts various oligomeric states in solution. Combined, these observations imply that the mechanism of membrane permeability by uperin 3.5 is related to its fibril-forming properties.Antonio N. Calabrese, Yanqin Liu, Tianfang Wang, Ian F. Musgrave, Tara L. Pukala, Rico F. Tabor, Lisandra L. Martin, John A. Carver, John H. Bowi
Optogenetics comes of age: novel inhibitory light-gated anionic channels allow efficient silencing of neural function

This is the peer reviewed version of the following article: A. Peralvárez-Marín, P. Garriga, Optogenetics Comes of Age: Novel Inhibitory Light-Gated Anionic Channels Allow Efficient Silencing of Neural Function. ChemBioChem 2016, 17, 204-206,  which has been published in final form at http://dx.doi.org/10.1002/cbic.201500608. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-ArchivingOptogenetics, the developing field of research that uses light-switchable biochemical tools in a sophisticated technological approach to monitor or control neural function, is rapidly evolving with the discovery and development of novel microbial rhodopsins. Light-absorbing membrane proteins, as tools for brain research, are promoting new applications within the discipline of optogenetics. Light-gated rhodopsin ion channels with better intrinsic light sensitivity and improved resolution are needed to overcome some of the current limitations of existing molecules. The recent discovery of light-gated inhibitory anion channels opens new opportunities for studying physiological neural processes and, at the same time, represent a powerful approach for elucidating the mechanisms of neurological and mental disorders that could benefit from this approach.Peer ReviewedPostprint (published version
Identification of Sequence Specificity of 5-Methylcytosine Oxidation by Tet1 Protein with High-Throughput Sequencing

Tet (ten-eleven translocation) family proteins have the ability to oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC). However, the oxidation reaction of Tet is not understood completely. Evaluation of genomic-level epigenetic changes by Tet protein requires unbiased identification of the highly selective oxidation sites. In this study, we used high-throughput sequencing to investigate the sequence specificity of mC oxidation by Tet1. A 6.6×10[4]-member mC-containing random DNA-sequence library was constructed. The library was subjected to Tet-reactive pulldown followed by high-throughput sequencing. Analysis of the obtained sequence data identified the Tet1-reactive sequences. We identified mCpG as a highly reactive sequence of Tet1 protein
Effect of ATRX and G-Quadruplex Formation by the VNTR Sequence on α-Globin Gene Expression

ATR-X (α-thalassemia/mental retardation X-linked) syndrome is caused by mutations in chromatin remodeler ATRX. ATRX can bind the variable number of tandem repeats (VNTR) sequence in the promoter region of the α-globin gene cluster. The VNTR sequence, which contains the potential G-quadruplex-forming sequence CGC(GGGGCGGGG)n, is involved in the downregulation of α-globin expression. We investigated G-quadruplex and i-motif formation in single-stranded DNA and long double-stranded DNA. The promoter region without the VNTR sequence showed approximately twofold higher luciferase activity than the promoter region harboring the VNTR sequence. G-quadruplex stabilizers hemin and TMPyP4 reduced the luciferase activity, whereas expression of ATRX led to a recovery in reporter activity. Our results demonstrate that stable G-quadruplex formation by the VNTR sequence downregulates the expression of α-globin genes and that ATRX might bind to and resolve the G-quadruplex
Chemical protein modification through cysteine

The modification of proteins with non-protein entities is important for a wealth of applications, and methods for chemically modifying proteins attract considerable attention. Generally, modification is desired at a single site to maintain homogeneity and to minimise loss of function. Though protein modification can be achieved by targeting some natural amino acid side chains, this often leads to ill-defined and randomly modified proteins. Amongst the natural amino acids, cysteine combines advantageous properties contributing to its suitability for site-selective modification, including a unique nucleophilicity, and a low natural abundanceboth allowing chemo- and regioselectivity. Native cysteine residues can be targeted, or Cys can be introduced at a desired site in a protein by means of reliable genetic engineering techniques. This review on chemical protein modification through cysteine should appeal to those interested in modifying proteins for a range of applications