Chemoenzymatic Late-Stage Modifications Enable Downstream Click-Mediated Fluorescent Tagging of Peptides

Aromatic prenyltransferases from cyanobactin biosynthetic pathways catalyse the chemoselective and regioselective intramolecular transfer of prenyl/geranyl groups from isoprene donors to an electron-rich position in these macrocyclic and linear peptides. These enzymes often demonstrate relaxed substrate specificity and are considered useful biocatalysts for structural diversification of peptides. Herein, we assess the isoprene donor specificity of the N1-tryptophan prenyltransferase AcyF from the anacyclamide A8P pathway using a library of 22 synthetic alkyl pyrophosphate analogues, of which many display reactive groups that are amenable to additional functionalization. We further used AcyF to introduce a reactive moiety into a tryptophan-containing cyclic peptide and subsequently used click chemistry to fluorescently label the enzymatically modified peptide. This chemoenzymatic strategy allows late-stage modification of peptides and is useful for many applications

Blind trials of computer-assisted structure elucidation software

<p>Abstract</p> <p>Background</p> <p>One of the largest challenges in chemistry today remains that of efficiently mining through vast amounts of data in order to elucidate the chemical structure for an unknown compound. The elucidated candidate compound must be fully consistent with the data and any other competing candidates efficiently eliminated without doubt by using additional data if necessary. It has become increasingly necessary to incorporate an <it>in silico </it>structure generation and verification tool to facilitate this elucidation process. An effective structure elucidation software technology aims to mimic the skills of a human in interpreting the complex nature of spectral data while producing a solution within a reasonable amount of time. This type of software is known as computer-assisted structure elucidation or CASE software. A systematic trial of the ACD/Structure Elucidator CASE software was conducted over an extended period of time by analysing a set of single and double-blind trials submitted by a global audience of scientists. The purpose of the blind trials was to reduce subjective bias. Double-blind trials comprised of data where the candidate compound was unknown to both the submitting scientist and the analyst. The level of expertise of the submitting scientist ranged from novice to expert structure elucidation specialists with experience in pharmaceutical, industrial, government and academic environments.</p> <p>Results</p> <p>Beginning in 2003, and for the following nine years, the algorithms and software technology contained within ACD/Structure Elucidator have been tested against 112 data sets; many of these were unique challenges. Of these challenges 9% were double-blind trials. The results of eighteen of the single-blind trials were investigated in detail and included problems of a diverse nature with many of the specific challenges associated with algorithmic structure elucidation such as deficiency in protons, structure symmetry, a large number of heteroatoms and poor quality spectral data.</p> <p>Conclusion</p> <p>When applied to a complex set of blind trials, ACD/Structure Elucidator was shown to be a very useful tool in advancing the computer's contribution to elucidating a candidate structure from a set of spectral data (NMR and MS) for an unknown. The synergistic interaction between humans and computers can be highly beneficial in terms of less biased approaches to elucidation as well as dramatic improvements in speed and throughput. In those cases where multiple candidate structures exist, ACD/Structure Elucidator is equipped to validate the correct structure and eliminate inconsistent candidates. Full elucidation can generally be performed in less than two hours; this includes the average spectral data processing time and data input.</p

K2-288Bb: A Small Temperate Planet in a Low-mass Binary System Discovered by Citizen Scientists

Original content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.Observations from the Kepler and K2 missions have provided the astronomical community with unprecedented amounts of data to search for transiting exoplanets and other astrophysical phenomena. Here, we present K2-288, a low-mass binary system (M2.0 ± 1.0; M3.0 ± 1.0) hosting a small (R p = 1.9 R ⊕), temperate (T eq = 226 K) planet observed in K2 Campaign 4. The candidate was first identified by citizen scientists using Exoplanet Explorers hosted on the Zooniverse platform. Follow-up observations and detailed analyses validate the planet and indicate that it likely orbits the secondary star on a 31.39-day period. This orbit places K2-288Bb in or near the habitable zone of its low-mass host star. K2-288Bb resides in a system with a unique architecture, as it orbits at >0.1 au from one component in a moderate separation binary (a proj ~ 55 au), and further follow-up may provide insight into its formation and evolution. Additionally, its estimated size straddles the observed gap in the planet radius distribution. Planets of this size occur less frequently and may be in a transient phase of radius evolution. K2-288 is the third transiting planet system identified by the Exoplanet Explorers program and its discovery exemplifies the value of citizen science in the era of Kepler, K2, and the Transiting Exoplanet Survey Satellite

Venomous gland transcriptome and venom proteomic analysis of the scorpion Androctonus amoreuxi reveal new peptides with anti-SARS-CoV-2 activity

The recent COVID-19 pandemic shows the critical need for novel broad spectrum antiviral agents. Scorpion venoms are known to contain highly bioactive peptides, several of which have demonstrated strong antiviral activity against a range of viruses. We have generated the first annotated reference transcriptome for the Androctonus amoreuxi venom gland and used high performance liquid chromatography, transcriptome mining, circular dichroism and mass spectrometric analysis to purify and characterize twelve previously undescribed venom peptides. Selected peptides were tested for binding to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and inhibition of the spike RBD – human angiotensin-converting enzyme 2 (hACE2) interaction using surface plasmon resonance-based assays. Seven peptides showed dose-dependent inhibitory effects, albeit with IC50 in the high micromolar range (117–1202 µM). The most active peptide was synthesized using solid phase peptide synthesis and tested for its antiviral activity against SARS-CoV-2 (Lineage B.1.1.7). On exposure to the synthetic peptide of a human lung cell line infected with replication-competent SARS-CoV-2, we observed an IC50 of 200 nM, which was nearly 600-fold lower than that observed in the RBD – hACE2 binding inhibition assay. Our results show that scorpion venom peptides can inhibit the SARS-CoV-2 replication although unlikely through inhibition of spike RBD – hACE2 interaction as the primary mode of action. Scorpion venom peptides represent excellent scaffolds for design of novel anti-SARS-CoV-2 constrained peptides. Future studies should fully explore their antiviral mode of action as well as the structural dynamics of inhibition of target virus-host interactions

Testing, assessment and literacy learning in schools: a view from England

Cyclic peptides are a class of compounds with high therapeutic potential, possessing bioactivities including antitumor and antiviral (including anti-HIV). Despite their desirability, efficient design and production of these compounds has not been achieved to date. The catalytic mechanism of patellamide macrocyclization by the PatG macrocyclase domain has been computationally investigated by using quantum mechanics/molecular mechanics methodology, specifically ONIOM(M06/6-311++G(2d,2p):ff94//B3LYP/6-31G(d):ff94). The mechanism proposed herein begins with a proton transfer from Ser783 to His 618 and from the latter to Asp548. Nucleophilic attack of Ser783 on the substrate leads to the formation of an acyl-enzyme covalent complex. The leaving group Ala-Tyr-Asp-Gly (AYDG) of the substrate is protonated by the substrate's N terminus, leading to the breakage of the P1-P1' bond. Finally, the substrate's N terminus attacks the P1 residue, decomposing the acyl-enzyme complex forming the macrocycle. The formation and decomposition of the acyl-enzyme complex have the highest activation free energies (21.1 kcal mol(-1) and 19.8 kcal mol(-1) respectively), typical of serine proteases. Understanding the mechanism behind the macrocyclization of patellamides will be important to the application of the enzymes in the pharmaceutical and biotechnological industries

Structure of the cyanobactin oxidase ThcOx from Cyanothece sp. PCC 7425, the first structure to be solved at Diamond Light Source beamline I23 by means of S-SAD.

Determination of protein crystal structures requires that the phases are derived independently of the observed measurement of diffraction intensities. Many techniques have been developed to obtain phases, including heavy-atom substitution, molecular replacement and substitution during protein expression of the amino acid methionine with selenomethionine. Although the use of selenium-containing methionine has transformed the experimental determination of phases it is not always possible, either because the variant protein cannot be produced or does not crystallize. Phasing of structures by measuring the anomalous diffraction from S atoms could in theory be almost universal since almost all proteins contain methionine or cysteine. Indeed, many structures have been solved by the so-called native sulfur single-wavelength anomalous diffraction (S-SAD) phasing method. However, the anomalous effect is weak at the wavelengths where data are normally recorded (between 1 and 2 Å) and this limits the potential of this method to well diffracting crystals. Longer wavelengths increase the strength of the anomalous signal but at the cost of increasing air absorption and scatter, which degrade the precision of the anomalous measurement, consequently hindering phase determination. A new instrument, the long-wavelength beamline I23 at Diamond Light Source, was designed to work at significantly longer wavelengths compared with standard synchrotron beamlines in order to open up the native S-SAD method to projects of increasing complexity. Here, the first novel structure, that of the oxidase domain involved in the production of the natural product patellamide, solved on this beamline is reported using data collected to a resolution of 3.15 Å at a wavelength of 3.1 Å. The oxidase is an example of a protein that does not crystallize as the selenium variant and for which no suitable homology model for molecular replacement was available. Initial attempts collecting anomalous diffraction data for native sulfur phasing on a standard macromolecular crystallography beamline using a wavelength of 1.77 Å did not yield a structure. The new beamline thus has the potential to facilitate structure determination by native S-SAD phasing for what would previously have been regarded as very challenging cases with modestly diffracting crystals and low sulfur content

Combination of Traction Assays and Multiphoton Imaging to Quantify Skin Biomechanics

International audienceAn important issue in tissue biomechanics is to decipher the relationship between the mechanical behavior at macroscopic scale and the organization of the collagen fiber network at microscopic scale. Here, we present a protocol to combine traction assays with multiphoton microscopy in ex vivo murine skin. This multiscale approach provides simultaneously the stress/stretch response of a skin biopsy and the collagen reorganization in the dermis by use of second harmonic generation (SHG) signals and appropriate image processing