Development of a peptide inhibitor-based cantilever sensor assay for cyclic adenosine monophosphate-dependent protein kinase

A highly sensitive nanomechanical cantilever sensor assay based on an electrical measurement has been developed for detecting activated cyclic adenosine monophosphate (cyclic AMP)-dependent protein kinase (PKA). Employing a peptide derived from the heat-stable protein kinase inhibitor (PKI), a magnetic bead system was first selected as a vehicle to immobilize the PKI-(5-24) peptide for capturing PKA catalytic subunit and the activity assay was applied for indirectly assessing the binding. Synergistic interactions of adenosine triphosphate (ATP) and the peptide inhibitor with the kinase were then investigated by a solution phase capillary electrophoretic assay, and by surface plasmon resonance technology which involved immobilization of the peptide inhibitor. After systemically evaluated by a homogeneous direct binding assay, the ATP-dependent recognition of the catalytic subunit of PKA by PKI-(5-24) was successfully transferred on to the nanomechanical cantilevers at protein concentrations of 6.6 pM-66 nM, exhibiting much higher sensitivity and wider dynamic range than the conventional activity assay. Thus, direct assessment of activated kinases using the cantilever sensor system functionalized with specific peptide inhibitors holds great promise in analytical applications and clinical medicine.     

Protein adsorption characteristics of calcium hydroxyapatites modified with pyrophosphoric acids

Protein adsorption characteristics of calcium hydroxyapatite (Hap) modified with pyrophosphoric acids (PP(a)) were examined. The PP(a) modified Hap particles (abbreviated as PP-Hap) possessed anchored polyphosphate (PP: P-{O-PO(OH)}(n)-OH) branches on their surfaces. The proteins of bovine serum albumin (BSA: isoelectric point (iep)=4.7, molecular mass (M(s))=67,200 Da, acidic protein), myoglobin (MGB: iep=7.0, M(s)=17,800 Da, neutral protein), and lysozyme (LSZ: iep=11.1, M(s)=14,600 Da, basic protein) were examined. The zeta potential (zp) of PP-Hap particles as a function of pH overlapped; zp-pH curves were independent of the concentration of pyrophosphoric acids (abbreviated as [PP(a)]) used for modifying Hap surface. The saturated amounts of adsorbed BSA (Delta n(ads)(BSA)) were increased three-fold by the surface modification with PP(a) though they were independent of the [PP(a)]. Furthermore, the fraction of BSA desorption was independent of the [PP(a)]. This enhancement of BSA adsorption onto the PP-Hap is due to the hydrogen bonding between oxygen and OH groups of the PP-branches and functional groups of BSA molecules. In the case of LSZ, a more higher adsorption enhancement was observed; the saturated amount of adsorbed LSZ (Delta n(ads)(LSZ)) for Hap modified at [PP(a)]=6 mmol/dm(3) was nine-fold than that for Hap unmodified. This remarkable adsorption enhancement was explained by a three-dimensional binding mechanism; LSZ molecules were trapped inside of the PP-branches. Hence, a fraction of LSZ desorption was decreased with an increase in the [PP(a)]; as more PP-branches are presented on the surface the higher retardation of LSZ desorption was induced. It was expected from their small size that MGB adsorb between the PP-branches as well as LSZ. However, the amounts of adsorbed MGB (Delta n(ads)(MGB)) did not vary and were independent of the [PP(a)] due to the small numbers of functional groups of MGB. In addition, no dependence of the fraction of MGB desorption on the [PP(a)] was observed. The results of zp for all the protein systems supported the mode of protein adsorption discussed. The anchored structure of the PP-branches developed on the Hap surface to provide three-dimensional protein adsorption spaces was proved by a comparative experiment that was elucidating the effect of pyrophosphate ions for BSA adsorption onto Hap.     

Magnetization Transfer via Residual Dipolar Couplings: Application to Proton–Proton Correlations in Partially Aligned Proteins

A novel three-dimensional NMR experiment is reported that allows the observation of correlations between amide and other protons via residual dipolar couplings in partially oriented proteins. The experiment is designed to permit quantitative measurement of the magnitude of proton–proton residual dipolar couplings in larger molecules and at higher degree of alignments. The observed couplings contain data valuable for protein resonance assignment, local protein structure refinement, and determination of low-resolution protein folds.     

Domain Analysis and Motif Matcher (DAMM): A Program to Predict Selectivity Determinants in Monosiga brevicollis PDZ Domains Using Human PDZ Data

Choanoflagellates are single-celled eukaryotes with complex signaling pathways. They are considered the closest non-metazoan ancestors to mammals and other metazoans and form multicellular-like states called rosettes. The choanoflagellate Monosiga brevicollis contains over 150 PDZ domains, an important peptide-binding domain in all three domains of life (Archaea, Bacteria, and Eukarya). Therefore, an understanding of PDZ domain signaling pathways in choanoflagellates may provide insight into the origins of multicellularity. PDZ domains recognize the C-terminus of target proteins and regulate signaling and trafficking pathways, as well as cellular adhesion. Here, we developed a computational software suite, Domain Analysis and Motif Matcher (DAMM), that analyzes peptide-binding cleft sequence identity as compared with human PDZ domains and that can be used in combination with literature searches of known human PDZ-interacting sequences to predict target specificity in choanoflagellate PDZ domains. We used this program, protein biochemistry, fluorescence polarization, and structural analyses to characterize the specificity of A9UPE9_MONBE, a M. brevicollis PDZ domain-containing protein with no homology to any metazoan protein, finding that its PDZ domain is most similar to those of the DLG family. We then identified two endogenous sequences that bind A9UPE9 PDZ with <100 μM affinity, a value commonly considered the threshold for cellular PDZ–peptide interactions. Taken together, this approach can be used to predict cellular targets of previously uncharacterized PDZ domains in choanoflagellates and other organisms. Our data contribute to investigations into choanoflagellate signaling and how it informs metazoan evolution.     

高蛋白食物—螺旋藻

螺旋藻为原核生物。其所含蛋白质的质量和数量均为植物世界之冠;尚含各种维生素和生长因子;细胞壁易被人体消化;风味可口;无毒性。     

毛细管电泳涂层柱技术的进展

毛细管电泳涂层柱是解决蛋白质在毛细管壁吸附的最有效的方法。较为系统地综述了毛细管电泳涂层柱的几种制作方法,指出了毛细管电泳涂层柱（包括毛细管电色谱柱）的发展趋势,３９篇。     

高效凝胶过滤色谱法分离测定豆薯种子蛋白

用高效凝胶蛋白往分离豆薯种子蛋白租提液,结合光电二极管阵列检测器对分离的蛋白峰进行紫外光谱扫描来确认蛋白的纯度,测定了3种蛋白的分子量,采用邻苯二甲醛（OPA）柱后衍生法测定了豆薯种子蛋白的氨基酸含量。     

The Relationship of Glutathione-S-Transferase and Multi-Drug Resistance-Related Protein 1 in Nitric Oxide (NO) Transport and Storage

Nitric oxide is a diatomic gas that has traditionally been viewed, particularly in the context of chemical fields, as a toxic, pungent gas that is the product of ammonia oxidation. However, nitric oxide has been associated with many biological roles including cell signaling, macrophage cytotoxicity, and vasodilation. More recently, a model for nitric oxide trafficking has been proposed where nitric oxide is regulated in the form of dinitrosyl-dithiol-iron-complexes, which are much less toxic and have a significantly greater half-life than free nitric oxide. Our laboratory has previously examined this hypothesis in tumor cells and has demonstrated that dinitrosyl-dithiol-iron-complexes are transported and stored by multi-drug resistance-related protein 1 and glutathione-S-transferase P1. A crystal structure of a dinitrosyl-dithiol-iron complex with glutathione-S-transferase P1 has been solved that demonstrates that a tyrosine residue in glutathione-S-transferase P1 is responsible for binding dinitrosyl-dithiol-iron-complexes. Considering the roles of nitric oxide in vasodilation and many other processes, a physiological model of nitric oxide transport and storage would be valuable in understanding nitric oxide physiology and pathophysiology.     

Two- and Three-Dimensional H/C PISEMA Experiments and Their Application to Backbone and Side Chain Sites of Amino Acids and Peptides

Two-dimensional ¹H/¹³C polarization inversion spin exchange at the magic angle experiments were applied to single crystal samples of amino acids to demonstrate their potential utility on oriented samples of peptides and proteins. High resolution is achieved and structural information obtained on backbone and side chain sites from these spectra. A triple-resonance experiment that correlates the ¹H–¹³C_α dipolar coupling frequency with the chemical shift frequencies of the α-carbon, as well as the directly bonded amide ¹⁵N site, is also demonstrated. In this experiment the large ¹H–¹³C_α heteronuclear dipolar interaction provides an independent frequency dimension that significantly improves the resolution among overlapping ¹³C resonances of oriented polypeptides, while simultaneously providing measurements of the ¹³C_α chemical shift, ¹H–¹³C dipolar coupling, and ¹⁵N chemical shift frequencies and angular restraints for backbone structure determination.     

Superslow Backbone Protein Dynamics as Studied by 1D Solid-State MAS Exchange NMR Spectroscopy

Superslow backbone dynamics of the protein barstar and the polypeptide polyglycine was studied by means of a solid-state MAS 1D exchange NMR method (time-reverse ODESSA) that can detect reorientation of nuclei carrying anisotropic chemical shift tensors. Experiments were performed on carbonyl 13C in polyglycine (natural abundance) and backbone 15N nuclei in uniformly 15N-enriched barstar within a wide range of temperatures in dry and wet powders for both samples. Two exchange processes were observed in the experiments: molecular reorientation and spin diffusion. Experimental conditions that are necessary to separate these two processes are discussed on a quantitative level. It was revealed that the wet protein undergoes molecular motion in the millisecond range of correlation times, whereas in dry protein and polyglycine molecular reorientations could not be detected. The correlation time of the motion in the wet barstar at room temperature is 50-100 ms; the activation energy is about 80 kJ/mol. Previously, protein motions with such a long correlation time could be observed only by methods detecting chemical exchange in solution (e.g., hydrogen exchange). The application of solid-state MAS exchange spectroscopy provides new opportunities in studying slow biomolecular dynamics that is important for the biological function of proteins.