ABSTRACT
Limitations ........................................................................................ 192 11.2 Expression and Purication of Cyclic Nucleotide-Free Sample .................. 193
11.2.1 Finding a Cyclic Nucleotide-Free Expression Host ......................... 193 11.2.2 Comparative Expression and Purication of PKG Iβ 92-363
from BL21(DE3) and TP2000 .......................................................... 194 11.2.3 Mass Spectrometry Analysis of Cyclic Nucleotide Contamination ..... 194 11.2.4 Isothermal Titration Calorimetry Analysis of PKG Iβ 92-363 ......... 196
11.3 Conclusions and Future Directions ............................................................... 197 Acknowledgments .................................................................................................. 198 References .............................................................................................................. 198
nucleotide-binding (CNB) domain is the most common and well characterized.4 CNB domains are ancient domains occurring in diverse multidomain proteins such as kinases,5 transcription factors,6-8 ion channels,9 nucleotidyl cyclases,10 guanine exchange factors,11 and acetyltransferases,12 often functioning as allosteric modulators of enzyme activity.13 These domains serve as conformational switches that structurally rearrange upon binding of the cyclic nucleotides. In mammalian physiology, cyclic nucleotides are critical second messengers that relay signals through these enzymes, regulating diverse physiological processes such as vision, learning and memory, vasodilation, and metabolism.1 Consistent with their importance in physiology, mutations in proteins that relay cyclic nucleotide signaling have been linked to a diverse cohort of diseases including achromatopsia,14,15 epilepsy,16 Carney complex,17,18 brolamellar hepatocellular carcinoma,19 thoracic aortic aneurysms, and acute aortic dissections.20 The universal importance of cyclic nucleotide signaling in the normal function of mammalian cells has stimulated decades of research into the CNB domain-containing proteins that relay these signals and rmly established some of these proteins as drug targets in the treatment of disease.21,22
The most well characterized mammalian receptors of cyclic nucleotides are the homologous cAMP-dependent (PKA) and cGMP-dependent protein kinases (PKG). PKA and PKG were initially puried from rabbit muscle and lobster tail muscle, respectively.23,24 Later, PKA was shown to consist of an R subunit and a C subunit, which tightly associate in the absence of cAMP, and dissociate upon cAMP binding to the R subunit, allowing the C subunit to phosphorylate downstream substrates.5 PKG similarly contains a regulatory (R) domain and a catalytic (C) domain that changes conformation in response to cGMP, although these are fused into a single polypeptide chain.
