Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.12 (PKG)
 2,515 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Selective detection of phosphopeptides from proteolytic digests is a challenging and highly relevant task in many proteomics applications. Often phosphopeptides are present in small amounts and need selective isolation or enrichment before identification. Here we report a novel automated method for the enrichment of phosphopeptides from complex mixtures. The method employs a two-dimensional column setup, with titanium oxide-based solid-phase material (Titansphere) as the first dimension and reversed-phase material as the second dimension. Phosphopeptides are separated from nonphosphorylated peptides by trapping them under acidic conditions on a TiO(2) precolumn. Nonphosphorylated peptides break through and are trapped on a reversed-phase precolumn after which they are analyzed by nanoflow LC-ESI-MS/MS. Subsequently, phosphopeptides are desorbed from the TiO(2) column under alkaline conditions, reconcentrated onto the reversed-phase precolumn, and analyzed by nanoflow LC-ESI-MS/MS. The selectivity and practicality of using TiO(2) precolumns for trapping phosphopeptides are demonstrated via the analysis of a model peptide RKISASEF, in a 1:1 mixture of a non- and a monophosphorylated form. A sample of 125 fmol of the phosphorylated peptide could easily be isolated from the nonphosphorylated peptide with a recovery above 90%. In addition, proteolytic digests of three different autophosphorylation forms of the 153-kDa homodimeric cGMP-dependent protein kinase are analyzed. From proteolytic digests of the fully autophosphorylated protein at least eight phosphorylation sites are identified, including two previously uncharacterized sites, namely, Ser-26 and Ser-44. Ser-26 is characterized as a minor phosphorylation site in purified PKG samples, while Ser-44 is identified as a novel in vitro autophosphorylation target. These results clearly show that TiO(2) has strong affinity for phosphorylated peptides, and thus, we conclude that this material has a high potential in the field of phosphoproteomics.
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PMID:Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. 1525 27

Nanoflow electrospray ionization time of flight mass spectrometry (ESI-TOF-MS) was used to study activation properties of the cGMP-dependent protein kinase (PKG). Our nanoflow ESI-TOF-MS analysis confirms that PKG mainly occurs as a 153 kDa homodimer and is able to bind four cGMP molecules, which is in agreement with the known stoichiometry. Binding order and stoichiometry of cGMP, the non-hydrolysable ATP analog beta,gamma-imidoadenosine 5'-triphosphate (AMPPNP) and Mn2+ for PKG were characterized as model for the active PKG-cGMP-ATP/Mg2+ complex. Already in the absence of cGMP, a noncovalent complex between PKG and two molecules of AMPPNP could be observed by ESI-TOF-MS. Binding of AMPPNP to PKG was strongly enhanced by the addition of MnCl2 to the spray solution. This is in agreement with binding of AMPPNP/Mn2+ in the ATP binding pocket of PKG since all protein kinases require a metal ion to accompany ATP in the ATP-binding pocket for proper positioning of the beta and gamma phosphates. Additionally, this finding could imply that within the inactive conformation of PKG, the autoinhibition-domain, when in contact with the substrate-docking domain, does not block the entrance to the ATP-binding site. In the presence of cGMP, less of the fully saturated PKG-(cGMP)4(AMPPNP/Mn2+)2 complex was observed, suggesting that the PKG-ATP interaction is weakened in the active conformation of PKG. Additionally, limited proteolysis in combination with native-ESI MS showed to be a useful tool to study the contact regions on the PKG-dimer and also allowed the rapid determination of the overall autophosphorylation status of the protein. These measurements indicated that autophosphorylation mainly occurs within the first 80 aminoterminal residues and involves in total 3-4 phosphates per subunit.
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PMID:Probing noncovalent protein-ligand interactions of the cGMP-dependent protein kinase using electrospray ionization time of flight mass spectrometry. 1546 51

The molecular mechanism of cGMP-dependent protein kinase activation by its allosteric regulator cyclic-3',5'-guanosine monophosphate (cGMP) has been intensely studied. However, the structural as well as thermodynamic changes upon binding of cGMP to type I cGMP-dependent protein kinase are not fully understood. Here we report a cGMP-induced shift of Gibbs free enthalpy (DeltaDeltaGD) of 2.5 kJ.mol-1 as determined from changes in tryptophan fluorescence using urea-induced unfolding for bovine PKG Ialpha. However, this apparent increase in overall stability specifically excluded the N-terminal region of the kinase. Analyses of tryptic cleavage patterns using liquid chromatography-coupled ESI-TOF mass spectrometry and SDS/PAGE revealed that cGMP binding destabilizes the N-terminus at the hinge region, centered around residue 77, while the C-terminus was protected from degradation. Furthermore, two recombinantly expressed mutants: the deletion fragment Delta1-77 and the trypsin resistant mutant Arg77Leu (R77L) revealed that the labile nature of the N-terminus is primarily associated with the hinge region. The R77L mutation not only stabilized the N-terminus but extended a stabilizing effect on the remaining domains of the enzyme as well. These findings support the concept that the hinge region of PKG acts as a stability switch.
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PMID:The hinge region operates as a stability switch in cGMP-dependent protein kinase I alpha. 1740 45