scholarly journals Expression des protéines VP2 et VP5 de la capside externe du virus de la fièvre catarrhale ovine

2009 ◽  
Vol 62 (2-4) ◽  
pp. 173
Author(s):  
F. Mohd Jaafar ◽  
H. Attoui ◽  
X. W. Li ◽  
O. Alpar ◽  
P. P.C. Mertens
Keyword(s):  
Cell Attachment ◽  
Animal Experiments ◽  
System Optimization ◽  
Bioinformatic Analysis ◽  
Virus Infectivity ◽  
Evolutionary Analysis ◽  
Live Attenuated Vaccine ◽  
X Ray ◽  
X Ray Crystallography ◽  
Mammalian Expression

Since 1998, nine bluetongue virus (BTV) strains from serotypes 1, 2, 4, 6, 8, 9 11, 16 and 25 have invaded Europe, killing more than two million animals (mainly sheep). Live attenuated vac­cines of BTV-2, 4, 9 and 16 have also been used in the region, in some cases causing further outbreaks of disease. Recent sequence analysis have shown that there have been cases of reassortment between field strains and live attenuated vaccine strains, hence the need for safer vaccines such as killed vaccines or recombi­nant protein-based subunit vaccines. The outer capsid protein VP2 of Culicoides-borne orbiviruses is the cell-attachment pro­tein, which carries sero-neutralization epitopes. We have cloned the open reading frame (ORF) (of segment 2) encoding VP2 and expressed the protein from BTV-4 in a bacterial expression sys­tem. The entire protein was found to be insoluble. Bioinformatic and evolutionary analysis showed that VP2 was composed of two ‘domains’, which were expressed separately using the same system. Optimization of expression conditions generated soluble proteins, indicating a correct fold of the expression products. These protein domains were used as antigens in mice experi­ments to raise antibodies against conformational epitopes. VP2 of BTV-4 was also expressed in a baculovirus system and was found to be soluble. The ORF encoding VP2 was cloned into a mammalian expression vector (pcDNA3.1) and is currently used (as a DNA vaccine) in animal experiments (using Chitosan as an adjuvant) to assess the antibody raising capacity of this formula­tion. One of the intended uses of this protein or its domains is to generate crystals for determination of the VP2 atomic structure using X-ray crystallography. VP5 of orbiviruses is a fusion pro­tein that is involved in membrane penetration during initiation of infection. The ORF (of genome segment 6) encoding VP5 of BTV-4 was cloned and expressed in the same bacterial system. Bioinformatic analysis also defined two domains of VP5. Only about 10% of the full length protein was soluble, while the two separated domains were over 90% soluble. Currently, VP5 and the two separate domains are being used in mice experiments to determine whether these can influence the immune response when concomitantly injected with the VP2 domains. VP5 was also cloned in pcDNA and used in mice experiments, formu­lated using Chitosan as an adjuvant. Antibodies collected from the mice reacted with the recombinant expressed VP5 showing high titres of antibodies. VP5 and its domains will also be used in X-ray crystallography. A plaque reduction assay was developed to determine whether the antibodies generated against VP2 domains or the VP2/VP5 mixture can neutralize virus infectivity in cell culture assays, opening the way for challenge assays in animals.

scholarly journals Neuropilin-1 is a host factor for SARS-CoV-2 infection

Science ◽  
2020 ◽  
Vol 370 (6518) ◽  
pp. 861-865 ◽  
Author(s):  
James L. Daly ◽  
Boris Simonetti ◽  
Katja Klein ◽  
Kai-En Chen ◽  
Maia Kavanagh Williamson ◽  
...  
Keyword(s):  
Cell Culture ◽  
Rna Interference ◽  
Cell Attachment ◽  
Host Factor ◽  
Terminal Sequence ◽  
X Ray ◽  
X Ray Crystallography ◽  
Neuropilin 1 ◽  
Host Cell Attachment ◽  
Carboxyl Terminal

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), uses the viral spike (S) protein for host cell attachment and entry. The host protease furin cleaves the full-length precursor S glycoprotein into two associated polypeptides: S1 and S2. Cleavage of S generates a polybasic Arg-Arg-Ala-Arg carboxyl-terminal sequence on S1, which conforms to a C-end rule (CendR) motif that binds to cell surface neuropilin-1 (NRP1) and NRP2 receptors. We used x-ray crystallography and biochemical approaches to show that the S1 CendR motif directly bound NRP1. Blocking this interaction by RNA interference or selective inhibitors reduced SARS-CoV-2 entry and infectivity in cell culture. NRP1 thus serves as a host factor for SARS-CoV-2 infection and may potentially provide a therapeutic target for COVID-19.

Difference Fourier Analysis of Glucose Embedded and Frozen Hydrated Purple Membrane

1982 ◽  
Vol 40 ◽  
pp. 74-75
Author(s):  
Jules S. Jaffe ◽  
Robert M. Glaeser
Keyword(s):  
Purple Membrane ◽  
Data Set ◽  
High Resolution Data ◽  
X Ray ◽  
X Ray Crystallography ◽  
Fourier Techniques ◽  
Versus Protein ◽  
The Difference ◽  
Difference Fourier ◽  
Ideal Method

Although difference Fourier techniques are standard in X-ray crystallography it has only been very recently that electron crystallographers have been able to take advantage of this method. We have combined a high resolution data set for frozen glucose embedded Purple Membrane (PM) with a data set collected from PM prepared in the frozen hydrated state in order to visualize any differences in structure due to the different methods of preparation. The increased contrast between protein-ice versus protein-glucose may prove to be an advantage of the frozen hydrated technique for visualizing those parts of bacteriorhodopsin that are embedded in glucose. In addition, surface groups of the protein may be disordered in glucose and ordered in the frozen state. The sensitivity of the difference Fourier technique to small changes in structure provides an ideal method for testing this hypothesis.

3-D reconstruction of molecular shape from microcrystal thin sections

1983 ◽  
Vol 41 ◽  
pp. 748-749
Author(s):  
S. Cusack ◽  
J.-C. Jésior
Keyword(s):  
Three Dimensional ◽  
Molecular Shape ◽  
Biological Molecules ◽  
Fourier Space ◽  
Single Particles ◽  
Thin Sections ◽  
Standard Methods ◽  
X Ray ◽  
X Ray Crystallography ◽  
Dimensional Reconstruction

Three-dimensional reconstruction techniques using electron microscopy have been principally developed for application to 2-D arrays (i.e. monolayers) of biological molecules and symmetrical single particles (e.g. helical viruses). However many biological molecules that crystallise form multilayered microcrystals which are unsuitable for study by either the standard methods of 3-D reconstruction or, because of their size, by X-ray crystallography. The grid sectioning technique enables a number of different projections of such microcrystals to be obtained in well defined directions (e.g. parallel to crystal axes) and poses the problem of how best these projections can be used to reconstruct the packing and shape of the molecules forming the microcrystal.Given sufficient projections there may be enough information to do a crystallographic reconstruction in Fourier space. We however have considered the situation where only a limited number of projections are available, as for example in the case of catalase platelets where three orthogonal and two diagonal projections have been obtained (Fig. 1).

Electron microscopy of rabbit FC crystals

1983 ◽  
Vol 41 ◽  
pp. 730-731
Author(s):  
Robert A. Grant ◽  
Laura L. Degn ◽  
Wah Chiu ◽  
John Robinson
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
High Resolution Electron Microscopy ◽  
Molecular Replacement ◽  
X Ray ◽  
X Ray Crystallography ◽  
Resolution Electron ◽  
Replacement Technique ◽  
Hydrated Crystal ◽  
Molecular Structure Analysis

Proteolytic digestion of the immunoglobulin IgG with papain cleaves the molecule into an antigen binding fragment, Fab, and a compliment binding fragment, Fc. Structures of intact immunoglobulin, Fab and Fc from various sources have been solved by X-ray crystallography. Rabbit Fc can be crystallized as thin platelets suitable for high resolution electron microscopy. The structure of rabbit Fc can be expected to be similar to the known structure of human Fc, making it an ideal specimen for comparing the X-ray and electron crystallographic techniques and for the application of the molecular replacement technique to electron crystallography. Thin protein crystals embedded in ice diffract to high resolution. A low resolution image of a frozen, hydrated crystal can be expected to have a better contrast than a glucose embedded crystal due to the larger density difference between protein and ice compared to protein and glucose. For these reasons we are using an ice embedding technique to prepare the rabbit Fc crystals for molecular structure analysis by electron microscopy.

Tele-Substitution Reactions in the Synthesis of a Promising Class of Antimalarials

2020 ◽  
Author(s):  
Marat Korsik ◽  
Edwin Tse ◽  
David Smith ◽  
William Lewis ◽  
Peter J. Rutledge ◽  
...  
Keyword(s):  
Heterocyclic Ring ◽  
Ring System ◽  
Substitution Reactions ◽  
Laboratory Notebook ◽  
Polar Solvents ◽  
X Ray ◽  
X Ray Crystallography ◽  
The Core ◽  
Nmr Data

<p></p><p>We have discovered and studied a <i>tele</i>substitution reaction in a biologically important heterocyclic ring system. Conditions that favour the <i>tele</i>-substitution pathway were identified: the use of increased equivalents of the nucleophile or decreased equivalents of base, or the use of softer nucleophiles, less polar solvents and larger halogens on the electrophile. Using results from X-ray crystallography and isotope labelling experiments a mechanism for this unusual transformation is proposed. We focused on this triazolopyrazine as it is the core structure of the <i>in vivo </i>active anti-plasmodium compounds of Series 4 of the Open Source Malaria consortium.</p> <p> </p> <p>Archive of the electronic laboratory notebook with the description of all conducted experiments and raw NMR data could be accessed via following link <a href="https://ses.library.usyd.edu.au/handle/2123/21890">https://ses.library.usyd.edu.au/handle/2123/21890</a> . For navigation between entries of laboratory notebook please use file "Strings for compounds in the article.pdf" that works as a reference between article codes and notebook codes, also this file contain SMILES for these compounds. </p><br><p></p>

Highly Electron-Deficient Pyridinium-Nitrones for Rapid and Tunable Inverse-Electron-Demand Strain-Promoted Alkyne-Nitrone Cycloaddition to Bicyclo[6.1.0]nonyne

2019 ◽  
Author(s):  
Praveen Gunawardene ◽  
Wilson Luo ◽  
Alexander M. Polgar ◽  
John F. Corrigan ◽  
Mark Workentin
Keyword(s):  
Density Functional Theory ◽  
Reaction Kinetics ◽  
Density Functional ◽  
Functional Theory ◽  
X Ray ◽  
Inverse Electron Demand ◽  
X Ray Crystallography ◽  
Electron Demand

<div> <div> <p>Highly accelerated inverse-electron-demand strain-promoted alkyne-nitrone cycloaddition (IED SPANC) between a sta- ble cyclooctyne (bicyclo[6.1.0]nonyne (BCN)) and nitrones delocalized into a Cα-pyridinium functionality is reported, with the most electron-deficient “pyridinium-nitrone” displaying among the most rapid cycloadditions to BCN that is currently reported. Density functional theory (DFT) and X-ray crystallography are explored to rationalize the effects of N- and Cα-substituent modifications at the nitrone on IED SPANC reaction kinetics and the overall rapid reactivity of pyridinium-delocalized nitrones.</p> </div> </div>

Simultaneous Generation of a [2×2] Grid-like Complex and a Linear Double Helicate: A Three-Level Self-Sorting Process

2019 ◽  
Author(s):  
Jean-François Ayme ◽  
Jean-Marie Lehn ◽  
Corinne Bailly ◽  
Lydia Karmazin
Keyword(s):  
Metal Complexes ◽  
Mass Spectroscopy ◽  
Metal Salts ◽  
Simultaneous Generation ◽  
X Ray ◽  
X Ray Crystallography ◽  
Sorting Process

<div>Two constitutional dynamic libraries (CDLs)—each containing two amines, two dialdehydes and two metal salts—have been found to self-sort, generating two pairs of imine-based metallosupramolecular architectures sharing no component, a [2×2] grid-like complex and a linear double helicate. These CDLs provided unique examples of a three-level self-sorting process, as only two imine-based ligand constituents, two metal complexes and two architectures were selected during their assembling out of all the possible combinations of their initial components. The metallosupramolecular architectures assembled were characterized by NMR, mass spectroscopy, and X-ray crystallography.</div>

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