Coexistence of para and ferromagnetic phases of Fe3+ in undoped CdZnTe (Zn r-v 4%) crystals

The signatures of the coexistence of para and ferromagnetic phases for the Fe3+ charge state of iron have been identified in the low temperature electron spin resonance (ESR) spectra in undoped CdZnTe (Zn ~ 4%) crystals and independently verified by superconducting quantum interference device (SQUID)and AC susceptibility measurements. In the paramagnetic phase the inverse of AC susceptibility follows the Curie-Weiss law. In the ferromagnetic phase the thermal evolution of magnetization follows the well-known Bloch T3/2 law. This is further supported by the appearance of hysteresis in the SQUID measurements at 2 Kbelow Tc which is expected to lie in between 2 and 2.5 K

Deciphering the Structural Basis of Eukaryotic Protein Kinase Regulation

<div><p>Eukaryotic protein kinases (EPKs) regulate numerous signaling processes by phosphorylating targeted substrates through the highly conserved catalytic domain. Our previous computational studies proposed a model stating that a properly assembled nonlinear motif termed the Regulatory (R) spine is essential for catalytic activity of EPKs. Here we define the required intramolecular interactions and biochemical properties of the R-spine and the newly identified “Shell” that surrounds the R-spine using site-directed mutagenesis and various <i>in vitro</i> phosphoryl transfer assays using cyclic AMP-dependent protein kinase as a representative of the entire kinome. Analysis of the 172 available Apo EPK structures in the protein data bank (PDB) revealed four unique structural conformations of the R-spine that correspond with catalytic inactivation of various EPKs. Elucidating the molecular entities required for the catalytic activation of EPKs and the identification of these inactive conformations opens new avenues for the design of efficient therapeutic EPK inhibitors.</p></div

The architecture of EPKs.

<p>(A) The conserved EPK structural core is shown mapped on the catalytic subunit of PKA (PDB ID: 1ATP). The N-lobe (Grey) is mostly composed of β-sheets and the C-lobe (tan) is mostly α-helical. ATP (black) and two atoms of magnesium (purple) are bound in the cleft between the lobes. (B) R-spine (maroon) and C-spine (yellow) are bound to the large αF-helix (black) in the center of the C-lobe. They span the whole kinase core and the C-spine is completed by the adenine ring of ATP (yellow). Activation loop phosphorylation at residue T197 (pT197 (red)) is crucial for the complete activation of PKA, and pT197 forms a H-bond with H87 (blue) in the αC-helix from the N-lobe. (C) The different components of the R-spine are labeled as RS1 from catalytic loop (tan), RS2 from the activation loop (tan), RS3 from the αC-helix (grey), RS4 from the β4(grey), and is anchored by RS0 (light blue) from the αF-helix (black). (D) A cartoon representation of the R-spine and the major components of the EPK core.</p

The R-spine and Shell configuration in the inactive state of EPKs.

<p>The four inactive conformations of the EPKs are shown with representative structures as well as cartoons in order to illustrate the configurations of the R-spine and Shell. The structures are inactive I (AKT; PDB ID: 1GZK), representing the DFG-out configuration; inactive II (Src; PDB ID: 1FMK), representing the C-helix out configuration; inactive III (AMPK; PDB ID: 3H4J), representing the HRD-out configuration; inactive IV (P38 MAPK; PDB ID: 1WFC), representing the twisted lobe configuration. The active EPK conformation (PKA; PDB ID: 1ATP) is shown for comparison (center).</p

Summary of the alignment of the more than 13,000 EPK sequences.

<p>Percentile of aromatic, aliphatic, hydrophobic (bold/italics), and representative amino acids for each R-spine and Shell residues from alignment of more than 13,000 EPK protein sequences.</p

Phosphorylation of the activation loop is involved in stabilizing the assembled R-spine.

<p>(A) A comparison of the phosphorylated (PDB ID: 1ATP) and unphosphorylated (PDB ID: 4DFY) structures of PKA showed that the H-bond between pT197 and H87 is disrupted in the unphosphorylated state and the two lobes move away from each other and twist. (B) The inactivation of the C-subunit by twisting, separation of the two lobes, and disruption of the H-bond is shown as a cartoon representation. (C) The hydrophilic R-spine mutants were coexpressed with PDK1 to ensure complete activation loop phosphorylation and their activity measured using a radioactive phosphoryl transfer assay. Then H87A mutation was introduced to the hydrophilic R-spine mutants and coexpressed with PDK1 and activity was measured.</p

Understanding the properties required for a catalytically active R-spine.

<p>PKA mutants were expressed in <i>E. coli</i> and the catalytic activity was analyzed using Western blot assay to determine the effect of (A) aromatic and aliphatic properties of the R-spine and (B) specific interactions of RS1. A qualitative Western blot assay (C) and quantitative radioactive phosphoryl transfer assay (D) were carried out for PKA mutants containing hydrophilic R-spine mutations. A qualitative Western blot assay (E) and quantitative radioactive phosphoryl transfer assay (F) were carried out for PKA mutants containing removal of side chain atoms of the R-spine residues. (G) Cartoon summary of the R-spine mutants (orange circle represents introducing a hydrophobic residue, red circle represents introducing a hydrophilic residue, and white circle represents removal of side chain atoms) along with a summary of their catalytic activity (green label indicates active and red label indicates inactive).</p

The role of the Shell for catalytic activity.

<p>(A) The 3-dimensional structure of the Shell (teal) is shown surrounding the R-spine (maroon) in PKA (PDB ID: 1ATP). (B) The R-spine and Shell are represented as a cartoon. (C) A radioactive phosphoryl transfer assay was carried out on various mutants elucidating the relation of the Shell with the R-spine and the required role for catalytic activity. (D) The catalytic activity of the PKA mutants is summarized as a cartoon representation for clarity (white circle represents removal of side chain atoms, green label represents active and red label represents inactive).</p