Using small-angle x-ray scattering, we have observed the cGMPinduced elongation of an active, cGMP-dependent, monomeric deletion mutant of cGMP-dependent protein kinase (⌬ 1-52 PKG-I␤). On saturation with cGMP, the radius of gyration of ⌬ 1-52 PKG-I␤ increases from 29.4 ؎ 0.1 Å to 40.1 ؎ 0.7 Å, and the maximum linear dimension increases from 90 Å ؎ 10% to 130 Å ؎ 10%. The elongation is due to a change in the interaction between structured regulatory (R) and catalytic (C) domains. A model of cGMP binding to ⌬ 1-52 PKG-I␤ indicates that elongation of ⌬ 1-52 PKG-I␤ requires binding of cGMP to the low-affinity binding site of the R domain. A comparison with cAMP-dependent protein kinase suggests that both elongation and activation require cGMP binding to both sites; cGMP binding to the low-affinity site therefore seems to be a necessary, but not sufficient, condition for both elongation and activation of ⌬ 1-52 PKG-I␤. We also predict that there is little or no cooperativity in cGMP binding to the two sites of ⌬ 1-52 PKG-I␤ under the conditions used here. Results obtained by using the ⌬ 1-52 PKG-I␤ monomer indicate that a previously observed elongation of PKG-I␣ is consistent with a pure change in the interaction between the R domain and the C domain, without alteration of the dimerization interaction. This study has revealed important features of molecular mechanisms in the biochemical network describing PKG-I␤ activation by cGMP, yielding new insight into ligand activation of cyclic nucleotide-dependent protein kinases, a class of regulatory proteins that is key to many cellular processes.C yclic guanosine monophosphate (cGMP) is a second messenger signaling molecule that is central to the regulation of many physiological processes, including smooth muscle tone, visual transduction, platelet aggregation, bone growth, and electrolyte and fluid homeostasis (see reviews in refs. 1-3). Its known targets are ion channels, which can alter cellular cation or anion transport; phosphodiesterases, which degrade cyclic nucleotides; and the cGMP-dependent protein kinases (PKGs), which are believed to be responsible for most intracellular cGMP actions. The PKG [first discovered by Kuo and Greengard (4)] is a serine͞threonine protein kinase whose substrates include ion channels and pumps, receptors, and enzymes that control intracellular Ca 2ϩ concentrations. In its active form, PKG can transfer the ␥-phosphate from ATP to target proteins, preferentially targeting serines in substrates having consensus sequence RRXSX, with some exceptions (1, 5). Phosphorylation of target proteins by PKG effects smooth muscle relaxation by reduction of cellular Ca 2ϩ . The PKGs and cAMP-dependent protein kinases (PKA) are homologous enzymes, and the two enzyme families share many similarities in biochemical function and domain organization.PKG is a dimer comprised of two identical monomers (1-3). Each monomer of PKG contains a regulatory domain (R) and a catalytic domain (C) on a single polypeptide chain. The monomers dimerize via interactions ...

Infrared (IR) spectroscopy of biological cells is a growing area of research, with many papers focusing on differences between the spectra of cancerous and noncancerous cells. Much of this research has been performed using a monolayer of dehydrated cells. We posit that the use of monolayers can introduce artefacts that lead to an apparent but inaccurate measurement of differences between cancerous and noncancerous cells. Additionally, the use of dried cells complicates the extraction of biochemical information from the IR spectra. We demonstrate that using suspensions of viable cells in aqueous suspension reduces measurement artefacts and facilitates determining the concentration of the major biochemical components via a linear least-squares fit of the component spectra to the spectrum of the cells.

No abstract