Functional studies with membrane-bound and detergent-solubilized α2-adrenergic receptors expressed in Sf9 cells

A chip-based biosensor technology using surface plasmon resonance (SPR) was developed for studying the interaction of ligands and G protein-coupled receptors (GPCRs). GPCRs, the fourth largest superfamily in the human genome, are the largest class of targets for drug discovery.We have expressed the three subtypes of α₂-adrenergic receptor (α₂-AR), a prototypical GPCR as functional fusion proteins in baculovirus-infected insect cells. The localization of the expressed receptor was observed in intracellular organelles, as detected by eGFP fluorescence. In addition, the deletion mutants of α_2B-AR, with a deletion in the 3rd intracellular loop, exhibited unaltered K_d values and enhanced stability, thus making them more promising candidates for crystallization. SPR demonstrated that small molecule ligands can bind the detergent-solubilized receptor, thus proving that α₂-AR is active even in a lipid-free environment. The K_d values obtained from the biosensor analysis and traditional ligand binding studies correlate well with each other. This is the first demonstration of the binding of a small molecule to the detergent-solubilized state of α₂-ARs and interaction of low-molecular mass-ligands in real time in a label-free environment. This technology will also allow the development of high throughput platform for screening a large number of compounds for generation of leads.     

Dynamic property is a key determinant for protein-protein interactions

Dynamic property is highly correlated with the biological functions of macromolecules, such as the activity and specificity of enzymes and the allosteric regulation in the signal transduction process. Applications of the dynamic property to protein function researches have been discussed and encouraging progresses have been achieved, for example, in enzyme activity and protein-protein docking studies. However, how the global dynamic property contributes to protein-protein interaction was still unclear. We have studied the dynamic property in protein-protein interactions based on Gaussian Network Model and applied it to classify biological and nonbiological protein-protein complexes in crystal structures. The global motion correlation between residues from the two protomers was found to be remarkably different for biological and nonbiological complexes. This correlation has been used to discriminate biological and nonbiological complexes in crystal and gave a classification rate of 86.9% in the cross-validation test. The innovation of this feature is that it is a global dynamic property which does not rely directly on the interfacial properties of the complex. In addition, the correlation of the global motions was found to be weakly correlated with the dissociation rate constant of protein complexes. We suggest that the dynamic property is a key determinant for protein-protein interaction, which can be used to discriminate native and crystal complexes and potentially be applied in protein-protein dynamic rate constants estimations.     

Resting Sinus Heart Rate and First Degree AV block: Modifiable Risk Predictors or Epiphenomena?

Simple and cost-effective tools that identify patients at increased risk for adverse cardiovascular events are actively sought. High resting sinus heart rate and first degree AV block are easily recognized and commonly encountered findings in a cardiology practice. A growing body of epidemiological and clinical evidence has been shown them to be independent predictors of all-cause and cardiovascular mortality, both in the general population and in patients with structural heart disease. This paper reviews the important role of heart rate and first degree AV block in predicting cardiovascular outcomes, examines the pathophysiological mechanisms underlying this increased risk, and discusses the effectiveness of available therapies to favorably modify these risk factors.     

Letting go: Deep computational modeling insights into pH-dependent calcium affinity

Calcium and other cofactors can feature as key additions to a molecular interface, to the extent that the cofactor is completely buried in the bound state. How can such an interaction be regulated then? The answer: By facilitating a switch through an allosteric network. Although a number of unbinding mechanisms are being characterized, an extensive computational study by Joswig et al. reveals a detailed model for the pattern recognition receptor langerin.     

Dimer Formation of a Stabilized Gβ1 Variant: A Structural and Energetic Analysis

In previous work, a strongly stabilized variant of the β1 domain of streptococcal protein G (Gβ1) was obtained by an in vitro selection method. This variant, termed Gβ1-M2, contains the four substitutions E15V, T16L, T18I, and N37L. Here we elucidated the molecular basis of the observed strong stabilizations. The contributions of these four residues were analyzed individually and in various combinations, additional selections with focused Gβ1 gene libraries were performed, and the crystal structure of Gβ1-M2 was determined. All single substitutions (E15V, T16L, T18I, and N37L) stabilize wild-type Gβ1 by contributions of between 1.6 and 6.0 kJ mol^− 1 (at 70 °C). Hydrophobic residues at positions 16 and 37 provide the major contribution to stabilization by enlarging the hydrophobic core of Gβ1. They also increase the tendency to form dimers, as shown by dependence on the concentration of apparent molecular mass in analytical ultracentrifugation, by concentration-dependent stability, and by a strongly increased van't Hoff enthalpy of unfolding. The 0.88-Å crystal structure of Gβ1-M2 and NMR measurements in solution provide the explanation for the observed dimer formation. It involves a head-to-head arrangement of two Gβ1-M2 molecules via six intermolecular hydrogen bonds between the two β strands 2 and 2′ and an adjacent self-complementary hydrophobic surface area, which is created by the T16L and N37L substitutions and a large 120° rotation of the Tyr33 side chain. This removal of hydrophilic groups and the malleability of the created hydrophobic surface provide the basis for the dimer formation of stabilized Gβ1 variants.     

X-ray crystal structure of Saccharomyces cerevisiae Pdx1 provides insights into the oligomeric nature of PLP synthases

The universal enzymatic cofactor vitamin B6 can be synthesized as pyridoxal 5-phosphate (PLP) by the glutamine amidotransferase Pdx1. We show that Saccharomyces cerevisiae Pdx1 is hexameric by analytical ultracentrifugation and by crystallographic 3D structure determination. Bacterial homologues were previously reported to exist in hexamer:dodecamer equilibrium. A small sequence insertion found in yeast Pdx1 elevates the dodecamer dissociation constant when introduced into Bacillus subtilis Pdx1. Further, we demonstrate that the yeast Pdx1 C-terminus contacts an adjacent subunit, and deletion of this segment decreases enzymatic activity 3.5-fold, suggesting a role in catalysis.

Structured summary

MINT-7147859: PDX1 (uniprotkb:P16451) and PDX1 (uniprotkb:P16451) bind (MI:0407) by cosedimentation in solution (MI:0028)MINT-7147899: PDX1 (uniprotkb:P37528) and PDX1 (uniprotkb:P37528) bind (MI:0407) by cosedimentation in solution (MI:0028)     

Carboxy group derivatization for enhanced electron‐transfer dissociation mass spectrometric analysis of phosphopeptides

A novel strategy based on carboxy group derivatization is presented for specific characterization of phosphopeptides. By tagging the carboxy group with 1‐(2‐pyrimidyl) piperazine (PP), the ion charge states of phosphopeptides can be largely enhanced, showing great advantages for sequencing phosphorylated peptides with electron‐transfer dissociation MS. Besides, after PP‐derivatization, most non‐specific bindings can be avoided by eliminating the interaction between the carboxy group and TiO₂, greatly improving the specificity of TiO₂‐based phosphopeptide enrichment strategy. Moreover, being tagged with a hydrophobic group, the retention time of phosphopeptides in RPLC can be prolonged, overcoming the difficulty of separating phosphopeptides in RPLC‐based approach. Together with several other advantages, such as ease of handling, rapid reaction time, broad applicability and good reproducibility, this PP‐derivatization method is promising for high‐throughput phosphoproteome research.     

绿盲蝽气味结合蛋白AlucOBP7的表达及气味结合特性

气味结合蛋白(odorant binding proteins, OBPs) 在昆虫嗅觉识别中起着重要的作用, 尤其是在运输外界脂溶性气味分子通过嗅觉感器淋巴液到达嗅觉受体(olfactory receptors, ORs)的过程中发挥关键作用。明确OBPs在昆虫同外界进行信息交流过程中的作用有利于阐明昆虫嗅觉识别的机制, 同时可为利用干扰昆虫嗅觉识别来进行害虫防治奠定理论基础。本研究克隆了一个绿盲蝽Apolygus lucorum (Meyer-Dür)气味结合蛋白AlucOBP7基因（GenBank登录号: JQ675724）, 并进行了原核表达, 以1-NPN为荧光探针采用荧光竞争结合实验研究了AlucOBP7蛋白和10种棉花挥发物及 6种性信息素类似物的结合能力。结果表明： 在10种棉花挥发物中, AlucOBP7能够和2 己酮及水杨酸甲酯有效结合, 结合常数分别为55.13 μmol/L和28.26 μmol/L。在6种盲蝽性信息素类似物中, 4-氧代-反-2-己烯醛和AlucOBP7 具有较强的结合能力, 结合常数为23.14 μmol/L。丁酸乙酯、 丁酸丁酯及己酸己酯也能够和AlucOBP7 有效结合, 但结合能力中等, 结合常数分别为30.58, 39.26和35.81 μmol/L。初步推测, AlucOBP7 可能是绿盲蝽性信息素结合蛋白(pheromone binding proteins, PBPs), 并在感受性信息素和植物挥发物的过程中发挥双重功能。     

An invertebrate [hydroxyproline]-modified neuropeptide: further evidence for a close evolutionary relationship between insect adipokinetic hormone and mammalian gonadotropin hormone family

An octapeptide of the adipokinetic hormone (AKH) peptide family is identified in the corpora cardiaca of the stink bug, Nezara viridula, by ESI-MS^N (electrospray ionization multistage MS). This is the second AKH in N. viridula and it has a hydroxyproline residue at position 6, whereas the major AKH (known as Panbo-RPCH) has Pro as the sixth amino acid residue. The correct sequence assignment of [Hyp⁶]-Panbo-RPCH is confirmed by retention time and MS spectra of the synthetic peptide. Various extraction procedures were followed to ascertain whether the hydroxylation is an artefact of extraction, or whether it is due to a true post-translational modification at the prohormone level. The proline hydroxylation is unique for invertebrate neuropeptides, while it has been described in the vertebrate gonadotropin-releasing hormone (GnRH). The current finding is another piece of evidence that AKH and GnRH form a peptide superfamily and are closely related evolutionarily. Biologically, [Hyp⁶]-Panbo-RPCH is active in vivo as an AKH, causing hyperlipaemia in the stink bug at low doses, indicating again that it is an endogenous, mature and functional hormone in this insect species.