Archaeal RNA polymerase: the influence of the protruding stalk in crystal packing and preliminary biophysical analysis of the Rpo13 subunit

2011 ◽  
Vol 39 (1) ◽  
pp. 25-30 ◽  
Author(s):  
Magdalena Wojtas ◽  
Bibiana Peralta ◽  
Marina Ondiviela ◽  
Maria Mogni ◽  
Stephen D. Bell ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Laser Light ◽  
Crystal Packing ◽  
Biophysical Characterization ◽  
Crystal Forms ◽  
Space Groups ◽  
Biophysical Analysis ◽  
Sulfolobus Shibatae ◽  
Archaeal Transcription

We review recent results on the complete structure of the archaeal RNAP (RNA polymerase) enzyme of Sulfolobus shibatae. We compare the three crystal forms in which this RNAP packs (space groups P212121, P21212 and P21) and provide a preliminary biophysical characterization of the newly identified 13-subunit Rpo13. The availability of different crystal forms for this RNAP allows the analysis of the packing degeneracy and the intermolecular interactions that determine this degeneracy. We observe the pivotal role played by the protruding stalk composed of subunits Rpo4 and Rpo7 in the lattice contacts. Aided by MALLS (multi-angle laser light scattering), we have initiated the biophysical characterization of the recombinantly expressed and purified subunit Rpo13, a necessary step towards the understanding of Rpo13's role in archaeal transcription.

scholarly journals Structural Insights of Three 2,4-Disubstituted Dihydropyrimidine-5-carbonitriles as Potential Dihydrofolate Reductase Inhibitors

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3286
Author(s):  
Lamya H. Al-Wahaibi ◽  
Althaf Shaik ◽  
Mohammed A. Elmorsy ◽  
Mohammed S. M. Abdelbaky ◽  
Santiago Garcia-Granda ◽  
...  
Keyword(s):  
Water Molecule ◽  
Dihydrofolate Reductase ◽  
Crystal Packing ◽  
Docking Simulation ◽  
X Ray Diffraction ◽  
Space Groups ◽  
Reductase Inhibitors ◽  
Inhibitory Potential ◽  
Human Dihydrofolate Reductase

In this report, we describe the structural characterization of three 2,4-disubstituted-dihydropyrimidine-5-carbonitrile derivatives, namely 2-{[(4-nitrophenyl)methyl]sulfanyl}-6-oxo-4-propyl-1,6-dihydropyrimidine-5-carbonitrile 1, 4-(2-methylpropyl)-2-{[(4-nitrophenyl)methyl]sulfanyl}-6-oxo-1,6-dihydropyrimidine-5-carbonitrile 2, and 2-[(2-ethoxyethyl)sulfanyl]-6-oxo-4-phenyl-1,6-dihydropyrimidine-5-carbonitrile monohydrate 3. An X-ray diffraction analysis revealed that these compounds were crystallized in the centrosymmetric space groups and adopt an L-shaped conformation. One of the compounds (3) crystallized with a water molecule. A cyclic motif (R22(8)) mediated by N–H···O hydrogen bond was formed in compounds 1 and 2, whereas the corresponding motif was not favorable, due to the water molecule, in compound 3. The crystal packing of these compounds was analyzed based on energy frameworks performed at the B3LYP/6-31G(d,p) level of theory. Various inter-contacts were characterized using the Hirshfeld surface and its associated 2D-fingerprint plots. Furthermore, a molecular docking simulation was carried out to assess the inhibitory potential of the title compounds against the human dihydrofolate reductase (DHFR) enzyme.

scholarly journals Characterization of an Amino-Terminal Dimerization Domain from Retroviral Restriction Factor Fv1

2006 ◽  
Vol 80 (16) ◽  
pp. 8225-8235 ◽  
Author(s):  
Kate N. Bishop ◽  
Gulnahar B. Mortuza ◽  
Steven Howell ◽  
Melvyn W. Yap ◽  
Jonathan P. Stoye ◽  
...  
Keyword(s):  
Leukemia Virus ◽  
Limited Proteolysis ◽  
Dimerization Domain ◽  
Biophysical Characterization ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Biophysical Analysis ◽  
Baculovirus System ◽  
Resistance To Infection

ABSTRACT The Fv1 protein is an endogenous factor in mice that confers resistance to infection by certain classes of murine leukemia virus, a phenomenon referred to as restriction. The mechanism of restriction is not understood, and the low endogenous level of Fv1 in cells has prevented any biochemical or biophysical analysis of the protein. We have now purified recombinant Fv1n protein from a baculovirus system and demonstrate that Fv1 exists in a multimeric form. Furthermore, we have mapped the position of two domains within the protein using limited proteolysis. Biophysical characterization of the N-terminal domain reveals that it comprises a highly helical and extended dimeric structure. Based on these biochemical and biophysical data, we propose a model for the arrangement of domains in Fv1 and suggest that dimerization of the N-terminal domain is necessary for Fv1 function to allow the protein to interact with multiple capsid protomers in retroviral cores.

scholarly journals Purification, crystallization and preliminary X-ray diffraction analysis of inner membrane complex (IMC) subcompartment protein 1 (ISP1) fromToxoplasma gondii

2012 ◽  
Vol 68 (7) ◽  
pp. 832-834 ◽  
Author(s):  
Michelle L. Tonkin ◽  
Shannon Brown ◽  
Josh R. Beck ◽  
Peter J. Bradley ◽  
Martin J. Boulanger
Keyword(s):  
Diffraction Analysis ◽  
X Ray Diffraction ◽  
Inner Membrane ◽  
Crystal Forms ◽  
X Ray ◽  
Space Groups ◽  
Membrane Complex ◽  
Specific Proteins ◽  
Inner Membrane Complex

The protozoan parasites of the Apicomplexa phylum are devastating global pathogens. Their success is largely due to phylum-specific proteins found in specialized organelles and cellular structures. The inner membrane complex (IMC) is a unique apicomplexan structure that is essential for motility, invasion and replication. The IMC subcompartment proteins (ISP) have recently been identified inToxoplasma gondiiand shown to be critical for replication, although their specific mechanisms are unknown. Structural characterization ofTgISP1 was pursued in order to identify the fold adopted by the ISPs and to generate detailed insight into how this family of proteins functions during replication. An N-terminally truncated form ofTgISP1 was purified fromEscherichia coli, crystallized and subjected to X-ray diffraction analysis. Two crystal forms ofTgISP1 belonging to space groupsP4132 orP4332 andP212121diffracted to 2.05 and 2.1 Å resolution, respectively.

scholarly journals Synthesis and biophysical analysis of modified thymine-containing DNA oligonucleotides

2017 ◽  
Vol 53 (8) ◽  
pp. 1389-1392 ◽  
Author(s):  
F. Kawasaki ◽  
P. Murat ◽  
Z. Li ◽  
T. Santner ◽  
S. Balasubramanian
Keyword(s):  
Biophysical Characterization ◽  
Dna Oligonucleotides ◽  
Biophysical Analysis

We report the synthesis of a 5-formyl-2′-deoxyuridine (5fU) phosphoramidite, and the preparation and biophysical characterization of oligonucleotides comprising all known, naturally observed eukaryotic thymidine modifications.

The low-resolution 3D-structure of porin, a channel-forming protein in E. coliouter membranes

1983 ◽  
Vol 41 ◽  
pp. 440-441
Author(s):  
A. Engel ◽  
D.L. Dorset ◽  
A. Massalski ◽  
J.P. Rosenbusch
Keyword(s):  
Crystal Packing ◽  
3D Structure ◽  
Gram Negative Bacteria ◽  
Protein Ratio ◽  
Crystal Forms ◽  
Large Molecules ◽  
Functional Studies ◽  
Voltage Dependent ◽  
Planar Membranes ◽  
Hexagonal Arrays

Porins represent a group of channel forming proteins that facilitate diffusion of small solutes across the outer membrane of Gram-negative bacteria, while excluding large molecules (>650 Da). Planar membranes reconstituted from purified matrix porin (OmpF protein) trimers and phospholipids have allowed quantitative functional studies of the voltage-dependent channels and revealed concerted activation of triplets. Under the same reconstitution conditions but using high protein concentrations porin aggregated to 2D lattices suitable for electron microscopy and image processing. Depending on the lipid-to- protein ratio three different crystal packing arrangements were observed: a large (a = 93 Å) and a small (a = 79 Å) hexagonal and a rectangular (a = 79 Å b = 139 Å) form with p3 symmetry for the hexagonal arrays. In all crystal forms distinct stain filled triplet indentations could be seen and were found to be morphologically identical within a resolution of (22 Å). It is tempting to correlate stain triplets with triple channels, but the proof of this hypothesis requires an analysis of the structure in 3 dimensions.

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