The regulatory subunit of PKA-I remains partially structured and undergoes β-aggregation upon thermal denaturation

Background: The regulatory subunit (R) of cAMP-dependent protein kinase (PKA) is a modular flexible protein that responds with large conformational changes to the binding of the effector cAMP. Considering its highly dynamic nature, the protein is rather stable. We studied the thermal denaturation of full-length RIα and a truncated RIα(92-381) that contains the tandem cyclic nucleotide binding (CNB) domains A and B. Methodology/Principal Findings: As revealed by circular dichroism (CD) and differential scanning calorimetry, both RIα proteins contain significant residual structure in the heat-denatured state. As evidenced by CD, the predominantly α-helical spectrum at 25°C with double negative peaks at 209 and 222 nm changes to a spectrum with a single negative peak at 212-216 nm, characteristic of β-structure. A similar α→β transition occurs at higher temperature in the presence of cAMP. Thioflavin T fluorescence and atomic force microscopy studies support the notion that the structural transition is associated with cross-β-intermolecular aggregation and formation of non-fibrillar oligomers. Conclusions/Significance: Thermal denaturation of RIα leads to partial loss of native packing with exposure of aggregation-prone motifs, such as the B' helices in the phosphate-binding cassettes of both CNB domains. The topology of the β-sandwiches in these domains favors inter-molecular β-aggregation, which is suppressed in the ligand-bound states of RIα under physiological conditions. Moreover, our results reveal that the CNB domains persist as structural cores through heat-denaturation. © 2011 Dao et al

Charge transport in lightly reduced graphene oxide: A transport energy perspective

10.1063/1.4792042Journal of Applied Physics1136-JAPI

Trap levels in graphene oxide: A thermally stimulated current study

10.1149/2.006302sslECS Solid State Letters22M17-M1

Coexistence of volatile and non-volatile resistive switching in 2D h-BN based electronic synapses

We present the first fabrication of electronic synapses using two dimensional (2D) hexagonal boron nitride (/j-BN) as active switching layer. The main advantage of these devices compared to the transition metal oxide (TMO) based counterparts is that multilayer h-BN stacks show both volatile and non-volatile resistive switching (RS) depending on the programming stresses applied, which allows implementing short-term (STP) and long-term plasticity (LTP) rules using a single device and without the need of complex architectures

Monitoring interactions between receptor tyrosine kinases and their downstream effector proteins in living cells using bioluminescence resonance energy transfer

ABSTRACT A limited number of whole-cell assays allow monitoring of receptor tyrosine kinase (RTK) activity in a signaling pathway-specific manner. We present the general use of the bioluminescence resonance energy transfer (BRET) technology to quantitatively study the pharmacology and signaling properties of the receptor tyrosine kinase (RTK) superfamily. RTK BRET-2 assays monitor, in living cells, the specific interaction between RTKs and their effector proteins, which control the activation of specific downstream signaling pathways. A total of 22 BRET assays have been established for nine RTKs derived from four subfamilies [erythroblastic leukemia viral (v-erb-b) oncogene homolog (ErbB), plateletderived growth factor (PDGF), neurotrophic tyrosine kinase receptor (TRK), vascular endothelial growth factor (VEGF)] monitoring the interactions with five effectors (Grb2, p85, Stat5a, Shc46, PLC␥1). These interactions are dependent on the RTK kinase activity and autophosphorylation of specific tyrosine residues in the carboxyl terminus. RTK BRET assays are highly sensitive for quantifying ligand-independent (constitutive), agonist-induced, or antagonist-inhibited RTK activity levels. We studied the signaling properties of the PDGF receptor, ␣ polypeptide (PDGFRA) isoforms (V561D; D842V and ⌬842-845) carrying activating mutations identified in gastrointestinal stromal tumors (GIST). All three PDGFRA isoforms are fully constitutively activated, insensitive to the growth factor PDGF-BB, but show differential sensitivity of their constitutive activity to be inhibited by the inhibitor imatinib (Gleevec). Epidermal growth factor receptor (EGFR) BRET structure-function studies identify the tyrosine residues 1068, 1114, and 1148 as the main residues mediating the interaction of EGFR with the adapter protein Grb2. The BRET technology provides an assay platform to study signaling pathway-specific RTK structurefunction and will facilitate drug discovery efforts for the identification of novel RTK modulators. Receptor tyrosine kinases (RTKs) represent a broad class of cell surface receptors that transduce signals across the cell membrane and regulate cell proliferation, survival, differentiation and migration □ S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: RTK, receptor tyrosine kinase; BRET, bioluminescence resonance energy transfer; PI3K, phosphatidyl inositol 3-kinase; PLC␥1, phospholipase C␥1; PKC, protein kinase C; STAT, signal transducer and activator of transcription; Luc, luciferase; GFP, green fluorescence protein; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; EGFR, epidermal growth factor receptor; PDGFRA, platelet-derived growth factor receptor, ␣ polypeptide; PBS, phosphate-buffered saline; BDNF, brain-derived neurotrophic factor; HEK, human embryonic kidney; GPCR, G protein-coupled receptor; FPRL1, formyl peptide receptor-like 1; VEGF, vascular endothelial growth factor; GIST, gastrointestinal stromal tumors; erlotinib, 4-(3-ethynylphenylamino)-6,7-bis(2-methoxyethoxy)quinazoline hydrochloride; imatinib, 4- 1440 assay