Expression, purification and crystallization of the catalytic subunit of protein kinase CK2 from Zea mays

1998 ◽  
Vol 54 (1) ◽  
pp. 143-145 ◽  
Author(s):  
Barbara Guerra ◽  
Karsten Niefind ◽  
Lorenzo A. Pinna ◽  
Dietmar Schomburg ◽  
Olaf-Georg Issinger
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Ck2 ◽  
Crystal Packing ◽  
Unit Cell ◽  
Monoclinic Space Group ◽  
X Rays ◽  
Α Subunit ◽  
Cell Parameters ◽  
Packing Parameter ◽  
Kinase Ck2

The catalytic (α) subunit of protein kinase CK2 (CK2α) was originally cloned and overexpressed in the Escherichia coli strain pT7-7/BL21(DE3). The protein has been purified to homogeneity and crystallized. The crystals belong to the monoclinic space group C2, they have unit-cell parameters a = 142.6, b = 61.3, c = 45.6 Å, β = 103.3° and diffract X-rays to at least 2.0 Å resolution. The calculated crystal packing parameter is Vm = 2.47 Å3 Da−1 suggesting that one CK2α molecule is contained in the asymmetric unit and that the solvent content of the unit cell is 50%.

Crystallization and preliminary X-ray diffraction analysis of the regulatory subunit of human protein kinase CK2

1999 ◽  
Vol 55 (4) ◽  
pp. 895-897 ◽  
Author(s):  
Laurent Chantalat ◽  
Didier Leroy ◽  
Odile Filhol ◽  
Nora Quitaine ◽  
Edmond M. Chambaz ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Ck2 ◽  
Unit Cell ◽  
Regulatory Subunit ◽  
Monoclinic Space Group ◽  
Unit Cell Parameters ◽  
Crystal Forms ◽  
Space Group ◽  
Cell Parameters ◽  
Kinase Ck2

Protein kinase CK2 is a tetramer composed of two α catalytic subunits and two β regulatory subunits. A C-terminal truncated form of the β subunit has been overproduced in Escherichia coli and purified to homogeneity. Two crystal forms of the truncated protein which diffract to at least 2 Å resolution have been obtained. Form I belongs to the monoclinic space group P21, with unit-cell parameters a = 49.9, b = 92.9, c = 53.7 Å, β = 96.3°, and yields plate-like crystals. Form II belongs to the tetragonal space group P42212, with unit-cell parameters a = 132.19, b = 132.19, c = 63.79 Å, and produces rod-shaped crystals. Both crystal forms have a functional dimer in the crystal asymmetric unit.

Interaction of tubulin and protein kinase CK2 in Trypanosoma equiperdum

2017 ◽  
Vol 72 (11-12) ◽  
pp. 459-465 ◽  
Author(s):  
Beatriz E. Boscán ◽  
Graciela L. Uzcanga ◽  
Maritza Calabokis ◽  
Rocío Camargo ◽  
Frank Aponte ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Ck2 ◽  
Polyacrylamide Gel Electrophoresis ◽  
Direct Interaction ◽  
Exchange Chromatography ◽  
Parasite Protein ◽  
Α Subunit ◽  
Trypanosoma Equiperdum ◽  
Kinase Ck2

AbstractA polypeptide band with an apparent molecular weight of 55,000 was phosphorylated in vitro in whole-cell lysates ofTrypanosoma equiperdum. This band corresponds to tubulin as demonstrated by immunoprecipitation of the phosphorylated polypeptide fromT. equiperdumextracts when anti-α and anti-β tubulin monoclonal antibodies were employed. A parasite protein kinase CK2 was in charge of modifying tubulin given that common mammalian CK2 inhibitors such as emodin and GTP, hindered the phosphorylation of tubulin and exogenously added casein. Interestingly, a divalent cation-dependent translocation of theT. equiperdumtubulin and the CK2 responsible for its phosphorylation was noticed, suggesting a direct interaction between these two proteins. Additionally, this fraction of tubulin and its kinase coeluted using separations based on parameters as different as charge (DEAE-Sepharose anion-exchange chromatography) and size (Sephacryl S-300 gel filtration chromatography). Analyses by non-denaturing polyacrylamide gel electrophoresis and immunoblot of the purified and radioactively labeled fraction containing both tubulin and the CK2 enzyme, established the phosphorylation of a single band that was recognized by anti-CK2 α-subunit and anti-tubulin antibodies. All these findings revealed a physical association between a pool of tubulin and a CK2 inT. equiperdum.

Crystallization and preliminary X-ray analysis of the unliganded recombinant catalytic subunit of cAMP-dependent protein kinase

1998 ◽  
Vol 54 (6) ◽  
pp. 1401-1404 ◽  
Author(s):  
Narendra Narayana ◽  
Pearl Akamine ◽  
Nguyen-Huu Xuong ◽  
Susan S. Taylor
Keyword(s):  
Protein Kinase ◽  
Unit Cell ◽  
Catalytic Subunit ◽  
Monoclinic Space Group ◽  
Dependent Protein Kinase ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

X-ray diffraction-quality crystals of the unliganded mouse recombinant catalytic subunit of cAMP-dependent protein kinase were grown by the hanging-drop vapour-diffusion technique using 2-methyl-2,4-pentanediol as precipitant. The crystals belong to the monoclinic space group P21 with unit-cell parameters a = 48.9, b = 147.4, c = 54.2 Å, β = 110.2°. A data set to 3.0 Å resolution with 92% completeness has been collected using synchrotron radiation. The unit cell contains four molecules of molecular weight 40 kDa with a corresponding volume solvent content of 45%.

Tight binding between a pool of the heterodimeric α/β tubulin and a protein kinase CK2 inTrypanosoma cruziepimastigotes

Parasitology ◽  
2005 ◽  
Vol 132 (4) ◽  
pp. 511-523 ◽  
Author(s):  
A. R. DE LIMA ◽  
R. MEDINA ◽  
G. L. UZCANGA ◽  
K. NORIS SUÁREZ ◽  
V. T. CONTRERAS ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Ck2 ◽  
Divalent Cations ◽  
Polyacrylamide Gel Electrophoresis ◽  
Tight Binding ◽  
Α Subunit ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Tubulin Antibodies ◽  
Kinase Ck2 ◽  
Β Tubulin

Tubulin is the predominant phosphoprotein inTrypanosoma cruziepimastigotes and is phosphorylated by a protein kinase CK2. Interestingly, the presence or absence of divalent cations affected the solubilization of a pool of the parasite tubulin and the CK2 responsible for its phosphorylation. This fraction of tubulin and its kinase co-eluted using phosphocellulose, DEAE-Sepharose and Sephacryl S-300 chromatographies. Anti-α tubulin antibodies co-immunoprecipitated both tubulin and the CK2 responsible for its phosphorylation, and anti-CK2 α-subunit antibodies immunoprecipitated radioactively labelled α and β tubulin from phosphorylated epimastigote homogenates. Additionally, native polyacrylamide gel electrophoresis of the purified and radioactively labelled fraction containing tubulin and its kinase demonstrated the phosphorylation of a unique band that reacted with both anti-CK2 α-subunit and anti-tubulin antibodies. Together, these results establish a strong interaction between a pool of the heterodimeric α/β tubulin and a CK2 in this parasite. Hydrodynamic measurements indicated that theT. cruzitubulin-CK2 complex is globular with an estimated size of 145·4–147·5 kDa.

scholarly journals Crystallographic and DFT Studies on Pyrrolo[1,2-c]imidazole Scaffolds

2014 ◽  
Vol 2014 ◽  
pp. 1-8
Author(s):  
Manikandan Jayaraman ◽  
Rajarathinam Balakrishnan ◽  
Kannan Muthu ◽  
Manivel Panneerselvam ◽  
Vasuki Gnanasambandam ◽  
...  
Keyword(s):  
Density Functional ◽  
Crystal Packing ◽  
Stable State ◽  
Chair Conformation ◽  
Unit Cell ◽  
Basis Set ◽  
Monoclinic Space Group ◽  
Cell Parameters ◽  
B3lyp Method ◽  
The Stability

The crystal structures of the compounds C15H14N4O2 (1) and C16H16N4O4 (2) are reported and analyzed by single crystal X-ray diffraction technique. Compounds (1) and (2) crystallized in monoclinic space group P21/c and Cc with four molecules in the unit cell, respectively. The unit cell parameters for compound (1) are a = 11.4501(15) Å, b = 9.7869(11) Å, c = 12.3653(15) Å, β = 90.997(11)°, and Volume = 1385.5(3) Å3 and for compound (2) are a = 13.865(2) Å, b = 6.9538(8) Å, c = 16.841(2) Å, β = 98.602(11)°, and Volume = 1605.4(4) Å3. In both compounds (1) and (2), the pyrrolidine ring adopts half-chair conformation. Moreover, both inter- and intramolecular N–H⋯O hydrogen bonds stabilize the crystal structure and play a crucial role in crystal packing. This intermolecular interaction alone constructs C11 chain motif in both compounds. It is also supported by weak intermolecular π-π interaction which is essential for the stability of the crystal packing. Further, the Density Functional Theory (B3LYP) method with standard 6-31G basis set was used in the calculation and calculated geometrical parameter is correlated with the corresponding experimental data. The obtained HOMO and LUMO energies are in negative values indicating that the compounds are in stable state.

scholarly journals Crystallization of the Ets1–Runx1–CBFβ–DNA complex formed on the TCRα gene enhancer

2014 ◽  
Vol 70 (10) ◽  
pp. 1380-1384 ◽  
Author(s):  
Masaaki Shiina ◽  
Keisuke Hamada ◽  
Taiko Inoue-Bungo ◽  
Mariko Shimamura ◽  
Shiho Baba ◽  
...  
Keyword(s):  
Crystal Packing ◽  
Cell Receptor ◽  
Unit Cell ◽  
X Rays ◽  
Unit Cell Parameters ◽  
Space Group ◽  
Cell Parameters ◽  
Intrinsically Disordered ◽  
Binding Domains ◽  
Gene Enhancer

Gene transcription is regulated in part through the assembly of multiple transcription factors (TFs) on gene enhancers. To enable examination of the mechanism underlying the formation of these complexes and their response to a phosphorylation signal, two kinds of higher-order TF–DNA assemblies were crystallized composed of an unmodified or phosphorylated Ets1 fragment, a Runx1(L94K) fragment and a CBFβ fragment on the T-cell receptor (TCR) α gene enhancer. Within these complexes, the Ets1 and Runx1 fragments contain intrinsically disordered regulatory regions as well as their DNA-binding domains. Crystals of the complex containing unmodified Ets1 belonged to space groupP212121, with unit-cell parametersa= 78.7,b= 102.1,c= 195.0 Å, and diffracted X-rays to a resolution of 2.35 Å, and those containing phosphorylated Ets1 belonged to the same space group, with unit-cell parametersa= 78.6,b= 101.7,c= 194.7 Å, and diffracted X-rays to a similar resolution. To facilitate crystallization, a Runx1 residue involved in a hydrophobic patch that was predicted to be engaged in crystal packing based on the previously reported structures of Runx1-containing crystals was mutated.

scholarly journals Biochemical evidence that the N-terminal segments of the α subunit and the β subunit play interchangeable roles in the activation of protein kinase CK2

FEBS Letters ◽  
1998 ◽  
Vol 441 (1) ◽  
pp. 29-33 ◽  
Author(s):  
Stefania Sarno ◽  
Oriano Marin ◽  
Paola Ghisellini ◽  
Flavio Meggio ◽  
Lorenzo A Pinna
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Ck2 ◽  
Β Subunit ◽  
Α Subunit ◽  
Biochemical Evidence ◽  
Kinase Ck2
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