Polarization colors of various purified collagens were studied in fibers of similar thickness. Three different soluble collagens of type I, insoluble collagen type I, lathyritic collagen type I, two p-N-collagens type I, pepsin extract collagen type II, two soluble collagens type III, p-N-collagen type III, and soluble collagen type V were submitted to a routine histopathologic procedure of fixation, preparation of 5-microns-thick sections, staining with Picrosirius red and examination under crossed polars. Polarization colors were determined for thin fibers (0.8 micron or less) an thick fibers, (1.6-2.4 microns). Most thin fibers of collagens and p-N-collagens showed green to yellowish-green polarization colors with no marked differences between the various samples. Thick fibers of all p-N-collagens, lathyritic and normal 0.15 M NaCl-soluble collagens showed green to greenish-yellow polarization colors, while in all other collagens, polarization colors of longer wavelengths (from yellowish-orange to red) were observed. These data suggested that fiber thickness was not the only factor involved in determining the polarization colors of Picrosirius red-stained collagens. Tightly packed and presumably, better aligned collagen molecules showed polarization colors of longer wavelengths. Thus, packing of collagen molecules and not only fiber thickness plays a role in the pattern of polarization colors of Picrosirius red-stained collagens.

The mammalian cAMP-dependent protein kinases have regulatory (R) subunits that show substantial homology in amino acid sequence with the catabolite gene activator protein (CAP), a cAMP-dependent gene regulatory protein from Escherichia coli. Each R subunit has two in-tandem cAMP binding domains, and the structure of each of these domains has been modeled by analogy with the crystal structure of CAP. Both the type I and II regulatory subunits have been considered, so that four cAMP binding domains have been modeled. The binding of cAMP in general is analogous in all the structures and has been correlated with previous results based on photolabeling and binding of cAMP analogues. The model predicts that the first cAMP binding domain correlates with the previously defined fast dissociation site, which preferentially binds N6-substituted analogues of cAMP. The second domain corresponds to the slow dissociation site, which has a preference for C8-substituted analogues. The model also is consistent with cAMP binding in the syn conformation in both sites. Finally, this model has targeted specific regions that are likely to be involved in interdomain contacts. This includes contacts between the two cAMP binding domains as well as contacts with the amino-terminal region of the R subunit and with the catalytic subunit.

Specificity for signaling by cAMP-dependent protein kinase (PKA) is achieved by both targeting and isoform diversity. The inactive PKA holoenzyme has two catalytic (C) subunits and a regulatory (R) subunit dimer (R 2 :C 2 ). Although the RIα, RIIα, and RIIβ isoforms are well studied, little is known about RIβ. We show here that RIβ is enriched selectively in mitochondria and hypothesized that its unique biological importance and functional nonredundancy will correlate with its structure. Small-angle X-ray scattering showed that the overall shape of RIβ 2 :C 2 is different from its closest homolog, RIα 2 :C 2 . The full-length RIβ 2 :C 2 crystal structure allows us to visualize all the domains of the PKA holoenzyme complex and shows how isoform-specific assembly of holoenzyme complexes can create distinct quaternary structures even though the R 1 :C 1 heterodimers are similar in all isoforms. The creation of discrete isoform-specific PKA holoenzyme signaling "foci" paves the way for exploring further biological roles of PKA RIβ and establishes a paradigm for PKA signaling.structural biology | signal transduction | allostery C yclic AMP-dependent protein kinase (PKA) regulates a plethora of biological events that extend from development and differentiation to memory, ion transport, and metabolism. The inactive PKA holoenzyme is composed of a regulatory (R) subunit dimer and two catalytic (C) subunits. Binding of cAMP to the R-subunits unleashes the C-subunits, thereby allowing phosphorylation of PKA substrates. Specificity of PKA signaling is achieved in large part by the isoform diversity of its R-subunits. There are two classes of R-subunits, RI and RII, which are subclassified into α and β subtypes. Each isoform is encoded by a unique gene and is functionally nonredundant. RIα-null mice display early embryonic lethality (1), whereas RIIβ-null mice have a lean phenotype and are resistant to diet-induced diabetes (2). Specific isoforms also are expressed differently in cells and tissues. The RIβ isoform is expressed abundantly in brain and spinal cord (3-5). Hippocampal slices from RIβ-null mice show a severe deficit in long-term potentiation and also in long-term depression and memory. Although RIα protein levels are increased in these mice, the hippocampal function is not rescued, suggesting a unique role for RIβ, which is the least studied Rsubunit (6, 7). Most localization studies have focused on the differences between RI and RII and showed that RI isoforms are diffused mainly in the cytoplasm, whereas RIIs typically are associated with membranous organelles. More than 50 A kinase-anchoring proteins (AKAPs) have been identified as targeting PKA to a specific subcellular location (8). The only potential AKAP that binds preferentially to the RIβ isoform identified so far is the neurofibromatosis 2 tumor suppressor protein, merlin (9).All R isoforms share the same domain organization, which includes a docking and dimerization (D/D) domain followed by an inhibitor sequence and two cAMP-binding domains (Fig. 1E)....

Venomous predatory animals, such as snakes, spiders, scorpions, sea anemones, and cone snails, produce a variety of highly stable cystine-constrained peptide scaffolds as part of their neurochemical strategy for capturing prey. Here we report a new family of four-cystine, three-loop conotoxins (designated framework 14). Three peptides of this family (flf14a-c) were isolated from the venom of Conus floridanus floridensis, and one (vil14a) was isolated from the venom of Conus villepinii, two worm-hunting Western Atlantic cone snail species. The primary structure for these peptides was determined using Edman degradation sequencing, and their cystine pairing was assessed by limited hydrolysis with a combination of CNBr and chymotrypsin under nonreducing, nonalkylating conditions in combination with MALDI-TOF MS analysis of the resulting peptidic fragments. CD spectra and nanoNMR spectroscopy of these conotoxins directly isolated from the cone snails revealed a highly helical secondary structure for the four conotoxins. Sequence-specific nanoNMR analysis at room temperature revealed a well-defined helix-loop-helix tertiary structure that resembles that of the Cs alpha/alpha scorpion toxins kappa-hefutoxin, kappa-KTx1.3, and Om-toxins, which adopt a stable three-dimensional fold where the two alpha-helices are linked by the two disulfide bridges. One of these conotoxins (vil14a) has a Lys/Tyr dyad, separated by approximately 6A, which is a conserved structural feature in K(+) channel blockers. The presence of this framework in scorpions and in cone snails indicates a common molecular imprint in the venom of apparently unrelated predatory animals and suggests a common ancestral genetic origin.