Conformational and Functional Effects Induced by D- and L-Amino Acid Epimerization on a Single Gene Encoded Peptide from the Skin Secretion of Hypsiboas punctatus

Skin secretion of Hypsiboas punctatus is the source of a complex mixture of bioactive compounds where peptides and small proteins prevail, similarly to many other amphibians. Among dozens of molecules isolated from H. punctatus in a proteomic based approach, we report here the structural and functional studies of a novel peptide named Phenylseptin (FFFDTLKNLAGKVIGALT-NH₂) that was purified as two naturally occurring D- and L-Phes configurations. The amino acid epimerization and C-terminal amidation for both molecules were confirmed by a combination of techniques including reverse-phase UFLC, ion mobility mass spectrometry, high resolution MS/MS experiments, Edman degradation, cDNA sequencing and solid-phase peptide synthesis. RMSD analysis of the twenty lowest-energy ¹H NMR structures of each peptide revealed a major 90° difference between the two backbones at the first four N-terminal residues and substantial orientation changes of their respective side chains. These structural divergences were considered to be the primary cause of the in vitro quantitative differences in antimicrobial activities between the two molecules. Finally, both molecules elicited equally aversive reactions in mice when delivered orally, an effect that depended entirely on peripheral gustatory pathways.     

Conformational Studies of Arabinoglucuronomannology (AGM)

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多肽诱导的大肠杆菌DegP(HtrA)蛋白酶的构象变化

具有分子伴侣和蛋白酶双重活性的大肠杆菌DegP蛋白,在热休克和其他应激条件下,对于降解和清除膜间质中变性或损伤的蛋白质起着十分重要的作用.到目前为止,已有几种蛋白质被鉴定出是DegP的天然底物.以前的研究表明,DegP的体内底物之一,PapG菌毛蛋白的羧基端多肽能够激活DegP的蛋白酶活性.然而这种激活的机制及生理意义均未见报道.用合成的PapG菌毛蛋白的羧基端多肽对这种激活的机制进行了初步研究.结果表明,DegP与多肽结合后发生了可检测的构象变化.圆二色性光谱结果显示,结合多肽后DegP的二级结构和三级结构均发生了一定的变化.凝胶排阻层析和动态光散射实验也揭示出DegP分子在一定程度上变小.进一步实验表明,DegP在多肽存在下,其疏水表面和催化位点均有所暴露.荧光各向异性结果显示出DegP在结合多肽后其构象柔性降低.对上述结果的意义进行了探讨.     

Efficient (18)F-Labeling of Large 37-Amino-Acid pHLIP Peptide Analogues and Their Biological Evaluation

Solid tumors often develop an acidic microenvironment, which plays a critical role in tumor progression and is associated with increased level of invasion and metastasis. The 37-residue pH (low) insertion peptide (pHLIP) is under study as an imaging platform because of its unique ability to insert into cell membranes at a low extracellular pH (pH(e) < 7). Labeling of peptides with [(18)F]-fluorine is usually performed via prosthetic groups using chemoselective coupling reactions. One of the most successful procedures involves the alkyne-azide copper(I) catalyzed cycloaddition (CuAAC). However, none of the known "click" methods have been applied to peptides as large as pHLIP. We designed a novel prosthetic group and extended the use of the CuAAC "click chemistry" for the simple and efficient (18)F-labeling of large peptides. For the evaluation of this labeling approach, a d-amino acid analogue of WT-pHLIP and an l-amino acid control peptide K-pHLIP, both functionalized at the N-terminus with 6-azidohexanoic acid, were used. The novel 6-[(18)F]fluoro-2-ethynylpyridine prosthetic group, was obtained via nucleophilic substitution on the corresponding bromo-precursor after 10 min at 130 °C with a radiochemical yield of 27.5 ± 6.6% (decay corrected) with high radiochemical purity ≥98%. The subsequent Cu(I)-catalyzed "click" reaction with the azido functionalized pHLIP peptides was quantitative within 5 min at 70 °C in a mixture of water and ethanol using Cu-acetate and sodium l-ascorbate. [(18)F]-d-WT-pHLIP and [(18)F]-l-K-pHLIP were obtained with total radiochemical yields of 5-20% after HPLC purification. The total reaction time was 85 min including formulation. In vitro stability tests revealed high stability of the [(18)F]-d-WT-pHLIP in human and mouse plasma after 120 min, with the parent tracer remaining intact at 65% and 85%, respectively. PET imaging and biodistribution studies in LNCaP and PC-3 xenografted mice with the [(18)F]-d-WT-pHLIP and the negative control [(18)F]-l-K-pHLIP revealed pH-dependent tumor retention. This reliable and efficient protocol promises to be useful for the (18)F-labeling of large peptides such as pHLIP and will accelerate the evaluation of numerous [(18)F]-pHLIP analogues as potential PET tracers.     

Experimental Studies and Dynamics Modeling Analysis of the Swimming and Diving of Whirligig Beetles (Coleoptera: Gyrinidae)

Whirligig beetles (Coleoptera, Gyrinidae) can fly through the air, swiftly swim on the surface of water, and quickly dive across the air-water interface. The propulsive efficiency of the species is believed to be one of the highest measured for a thrust generating apparatus within the animal kingdom. The goals of this research were to understand the distinctive biological mechanisms that allow the beetles to swim and dive, while searching for potential bio-inspired robotics applications. Through static and dynamic measurements obtained using a combination of microscopy and high-speed imaging, parameters associated with the morphology and beating kinematics of the whirligig beetle''s legs in swimming and diving were obtained. Using data obtained from these experiments, dynamics models of both swimming and diving were developed. Through analysis of simulations conducted using these models it was possible to determine several key principles associated with the swimming and diving processes. First, we determined that curved swimming trajectories were more energy efficient than linear trajectories, which explains why they are more often observed in nature. Second, we concluded that the hind legs were able to propel the beetle farther than the middle legs, and also that the hind legs were able to generate a larger angular velocity than the middle legs. However, analysis of circular swimming trajectories showed that the middle legs were important in maintaining stable trajectories, and thus were necessary for steering. Finally, we discovered that in order for the beetle to transition from swimming to diving, the legs must change the plane in which they beat, which provides the force required to alter the tilt angle of the body necessary to break the surface tension of water. We have further examined how the principles learned from this study may be applied to the design of bio-inspired swimming/diving robots.     

Familial translocation t(10;21)(q22;q22)

Summary A family is described with a translocation t(10;21)(q22;q22) transmitted through three generations. This family was studied for the apparition of several miscarriages and two sisters with multiple malformations. Both children had a probably partial trisomy of chromosome 10 and a monosomy of chromosome 21 due to a maternal adjacent-2 meiotic segregation.     

Solution Structural Studies of GTP:Adenosylcobinamide-Phosphateguanylyl Transferase (CobY) from Methanocaldococcus jannaschii

GTP:adenosylcobinamide-phosphate (AdoCbi-P) guanylyl transferase (CobY) is an enzyme that transfers the GMP moiety of GTP to AdoCbi yielding AdoCbi-GDP in the late steps of the assembly of Ado-cobamides in archaea. The failure of repeated attempts to crystallize ligand-free (apo) CobY prompted us to explore its 3D structure by solution NMR spectroscopy. As reported here, the solution structure has a mixed α/β fold consisting of seven β-strands and five α-helices, which is very similar to a Rossmann fold. Titration of apo-CobY with GTP resulted in large changes in amide proton chemical shifts that indicated major structural perturbations upon complex formation. However, the CobY:GTP complex as followed by ¹H-¹⁵N HSQC spectra was found to be unstable over time: GTP hydrolyzed and the protein converted slowly to a species with an NMR spectrum similar to that of apo-CobY. The variant CobY^G153D, whose GTP complex was studied by X-ray crystallography, yielded NMR spectra similar to those of wild-type CobY in both its apo- state and in complex with GTP. The CobY^G153D:GTP complex was also found to be unstable over time.     

Binding,Conformational Transition and Dimerization of Amyloid-β Peptide on GM1-Containing Ternary Membrane: Insights from Molecular Dynamics Simulation

Interactions of amyloid-β (Aβ) with neuronal membrane are associated with the progression of Alzheimer’s disease (AD). Ganglioside GM1 has been shown to promote the structural conversion of Aβ and increase the rate of peptide aggregation; but the exact nature of interaction driving theses processes remains to be explored. In this work, we have carried out atomistic-scale computer simulations (totaling 2.65 µs) to investigate the behavior of Aβ monomer and dimers in GM1-containing raft-like membrane. The oligosaccharide head-group of GM1 was observed to act as scaffold for Aβ-binding through sugar-specific interactions. Starting from the initial helical peptide conformation, a β-hairpin motif was formed at the C-terminus of the GM1-bound Aβ-monomer; that didn’t appear in absence of GM1 (both in fluid POPC and liquid-ordered cholesterol/POPC bilayers and also in aqueous medium) within the simulation time span. For Aβ-dimers, the β-structure was further enhanced by peptide-peptide interactions, which might influence the propensity of Aβ to aggregate into higher-ordered structures. The salt-bridges and inter-peptide hydrogen bonds were found to account for dimer stability. We observed spontaneous formation of intra-peptide D²³-K²⁸ salt-bridge and a turn at V²⁴GSN²⁷ region - long been accepted as characteristic structural-motifs for amyloid self-assembly. Altogether, our results provide atomistic details of Aβ-GM1 and Aβ-Aβ interactions and demonstrate their importance in the early-stages of GM1-mediated Aβ-oligomerisation on membrane surface.     

A_（8—10）替换的胰岛素类似物的合成及生物活性

本文报道了用Ｆｍｏｃ固相法合成３种胰岛素Ａ链小环（Ａ８－１０）被不同碱性氨基酸取代的Ａ链类似物,并分别与天然胰岛素Ｂ链重组成相应胰岛素类似物;经受体结合,整体活性及抗体结合实验,均表现出相应的活性。从中可以推测出：Ａ链小环区域不是胰岛素表现生物活性的重要部位,而是胰岛素与其抗体结合较重要的区域。     

Conformational Flexibility of the Peptide Hormone Ghrelin in Solution and Lipid Membrane Bound: A Molecular Dynamics Study

Abstract

Human ghrelin is a peptide hormone of 28 aminoacid residues, in which the Ser3 is modified by an octanoyl group. Ghrelin has a major role in the energy metabolism of the human body stimulating growth hormone release as well as food intake. Here we perform molecular dynamics simulations in explicit water and in a DMPC-lipid bilayer/water system in order to structurally characterize this highly flexible peptide and its lipid binding properties. We find a loop structure with residues Glu17 to Lys 20 in the bending region and a short α-helix from residues Pro7 to Glu13. The presence of a lipid membrane does not influence these structural features, but reduces the overall flexibility of the molecule as revealed by reduced root mean square fluctuations of the atom coordinates. The octanoyl-side chain does not insert into the lipid membrane but points into the water phase. The peptide binds to the lipid membrane with its bending region involving residues Arg15, Lys16, Glu17, and Ser18. The implications of these results for the binding pocket of the ghrelin receptor are discussed.     

Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry Enables Multiplex,Quantitative Pharmacodynamic Studies of Phospho-Signaling

In most cell signaling experiments, analytes are measured one Western blot lane at a time in a semiquantitative and often poorly specific manner, limiting our understanding of network biology and hindering the translation of novel therapeutics and diagnostics. We show the feasibility of using multiplex immuno-MRM for phospho-pharmacodynamic measurements, establishing the potential for rapid and precise quantification of cell signaling networks. A 69-plex immuno-MRM assay targeting the DNA damage response network was developed and characterized by response curves and determinations of intra- and inter-assay repeatability. The linear range was ≥3 orders of magnitude, the median limit of quantification was 2.0 fmol/mg, the median intra-assay variability was 10% CV, and the median interassay variability was 16% CV. The assay was applied in proof-of-concept studies to immortalized and primary human cells and surgically excised cancer tissues to quantify exposure–response relationships and the effects of a genomic variant (ATM kinase mutation) or pharmacologic (kinase) inhibitor. The study shows the utility of multiplex immuno-MRM for simultaneous quantification of phosphorylated and nonmodified peptides, showing feasibility for development of targeted assay panels to cell signaling networks.Because there is limited correlation between mRNA and protein levels/activity (1), quantification of proteins and post-translational modifications is critical to understanding cellular signaling and determining pharmacodynamic (PD)¹ responses. Phosphorylation is a key post-translational modification used in signaling networks to modulate protein/pathway activity, protein interactions, and protein localization in response to extracellular and intracellular stimuli. Many diseases exhibit dysfunctions in signaling networks, and thus major efforts to identify novel drug targets (e.g. kinase inhibitors) are based on signal transduction pathways (2).Currently the research community lacks high throughput, quantitative tools for studying phospho-signaling networks, hindering our basic understanding of network biology and hence the translation of novel therapeutics and companion diagnostics. In most experiments, one analyte is measured one Western blot lane at a time in a semiquantitative and often nonspecific manner. These drawbacks limit our ability to extend knowledge beyond individual phosphorylation events to a system-wide study of phosphorylation dynamics, which is critical because signal transduction pathways act as interconnected networks, and the effects of mutations in individual genes (as well as the effects of pharmacologic compounds) spread throughout the network (3). Although Western blotting and related traditional immuno-assay platforms (e.g. ELISA) have been pushed brilliantly to their limits and have formed the basis of many advances in biomedical research, they are inadequate to support the needs of the postgenomic world, in which we need innovative technologies for determining the effects of any experimental condition (e.g. agonist or antagonist exposures, genetic variations) on the major signal transduction networks of the human cell, using precise, standardized, moderate-to-high throughput methods that can be reproduced across laboratories.Newer technologies, such as planar (4) or bead-based protein arrays (5) and mass cytometry (6) have shown potential for improving our ability to quantify signaling networks. However, like traditional immunoassays, these techniques do not directly detect and quantify the target analyte. Rather, the concentration of the target is inferred from a reporter signal, such as a fluorescent or mass tag on the antibody. As a result, these assay platforms are plagued by interferences present in biological matrices, which undermine the specificity of the assays in all but the most rigorously optimized settings using highly monospecific antibodies (7). Thus, generating such assays and assuring specificity is costly, time-consuming, and very difficult, especially in multiplex.Technological advancements in MS have enabled an impressive depth of coverage of the phosphoproteome using untargeted (“shotgun”) approaches (8–10). Furthermore, shotgun mass spectrometry has been used to profile signaling pathways by enriching phosphorylated peptides through approaches such as antiphospho-tyrosine antibodies (11), extensive fractionation (12), or panels of antibodies to enrich for signaling nodes (13). Coupling isotopic labeling methods (14, 15) to MS allows relative quantification of detectable peptides between two or a small number of samples, but these methods do not provide the absolute abundances of the peptides detected, nor are they amenable to the analysis of large numbers of biological samples. For example, to achieve substantial depth of coverage, multidimensional biochemical fractionations are required (9), limiting the number of samples that can be analyzed. Relatively large sample consumption is a constraint for analyzing clinical specimens. Under-sampling remains an issue in data-dependent modes, and missing information in the data is substantial. Thus, untargeted mass spectrometry is capable of broad discovery, but does not have adequate throughput or reproducibility for more expansive biological or clinical studies.There has been tremendous growth in the application of targeted quantitative MS to quantify proteotypic peptides (16–19). In contrast to untargeted “shotgun” modes of MS, targeted MS focuses the full analytic capacity of the instrument on selected analytes of interest, moving from a stochastic sampling of the complex mixture where the instrument “decides” (nonreproducibly and with low precision) what is analyzed- to targeted, high-precision measurements of suites of proteins of interest that can be context-dependent based on the biological question being asked. The most widely used form of targeted MS is multiple reaction monitoring (MRM). MRM has been the clinical gold standard for decades in pharmaceutical research and in clinical reference laboratories for quantification of small molecules such as drug metabolites or metabolites that accumulate as a result of inborn errors of metabolism (20, 21). Although proteolysis of the biospecimen is a source of preanalytical variation for peptide quantification, careful selection of peptide analytes readily produces MRM-based assays of high precision (%CV ≤ 20%) (17, 22) that enable accurate quantification of reproducibly released tryptic peptides. Also, because MRM assays use internal standards (i.e. synthetic, stable isotope-labeled peptides that are spiked into the biospecimen), assays yield highly reproducible results when shared among laboratories and implemented on different instrument platforms (23), even at high multiplex levels on an international stage (22). The molecular specificity of MRM is very high, as it is conferred by three orthogonal physiochemical properties of each peptide: its mass, its retention properties on HPLC, and the production of a set of fragment ions of specific mass (of which three to five are usually monitored) detected at characteristic ratios. High specificity, coupled to the large linear range of MRM (>10³) and the ability to monitor analytes at scheduled times during the MRM run, render MRM assays highly multiplexable. A recent study shows scalability of MRM via analytical validation of four multiplex MRM assays (ranging from 156–169 plex) quantifying 645 human peptides (319 proteins) with median %CV<6 and successful reproduction of assay results internationally across three laboratories (22). Thus, MRM shows many advantages over alternative protein measurement technologies, including the capability to multiplex with ease, the use of internal standards (aiding reproducible quantification and cross-laboratory standardization), high specificity through direct measurement of the analyte, and relatively less time and cost associated with assay development.Although immuno-MRM has previously been applied to quantify unmodified protein abundances in body fluids (24, 25), cell lysates, and tissues (26), the feasibility and success rate for using immuno-MRM to quantify phosphopeptides and phospho-signaling has not been tested. In this study, we tested the possibility that MRM could be coupled to peptide immuno-affinity enrichment (27, 28) to enable multiplex quantification of phospho-signaling networks. As proof-of-concept, we developed a 69-plex immuno-MRM assay for quantifying phospho-signaling in the DNA damage response (DDR) network. The DDR is critical for maintaining genomic integrity, and mutations in the DDR are among the most frequently identified in tumors (29). In this study we show: (1) simultaneous analysis of modified and nonmodified isoforms of peptides, (2) multiplexed quantitative analysis of signaling events, and (3) applicability to a variety of sample types and conditions. The multiplexed assay presented in this study replaces 69 Western blots with a 40 min MRM-MS run, providing quantitative, precise, specific data, and establishing feasibility for developing targeted assay panels to many cell signaling networks. The ability to quantitatively measure a large array of phosphorylation events would have broad benefits for the biomedical research community, spanning fundamental biological studies through to PD characterizations of novel drug compounds and the development of companion diagnostics.     

Synthesis,Spectral and Antibacterial Studies of Binuclear Titanium(IV) / Zirconium(IV) Complexes of Piperazine Dithiosemicarbazones

The reactions of mono(cyclopentadienyl)titanium(IV) trichloride and bis(cyclopentadienyl)titanium(IV)/ zirconium(IV) dichloride with a new class of dithiosemicarbazone, derived by condensing piperazine dithiosemicarbazide with benzaldehyde (L₁H₂), 2-chlorobenzaldehyde (L₂H₂), 4-nitrobenzaldehyde (L₃H₂) or salicylaldehyde (L₄H₄) have been studied and different types of binuclear products, viz. [{CpTiCl₂}₂L], [{Cp₂MCl}₂L], ((L=L₁, L₂ or L₃), [{CpTiCI}₂L₄] and [{Cp₂M}₂L₄] (M=Yi or Zr), have been isolated. Tentative structures are proposed for these complexes based upon elemental analyses, electrical conductance, magnetic moment and spectral (electronic, IR, ¹H and ¹³C NMR) data. Attempts have been made to establish a correlation between antibacterial activity and the structures of the products.     

Conformational Changes and Slow Dynamics through Microsecond Polarized Atomistic Molecular Simulation of an Integral Kv1.2 Ion Channel

Structure and dynamics of voltage-gated ion channels, in particular the motion of the S4 helix, is a highly interesting and hotly debated topic in current membrane protein research. It has critical implications for insertion and stabilization of membrane proteins as well as for finding how transitions occur in membrane proteins—not to mention numerous applications in drug design. Here, we present a full 1 µs atomic-detail molecular dynamics simulation of an integral Kv1.2 ion channel, comprising 120,000 atoms. By applying 0.052 V/nm of hyperpolarization, we observe structural rearrangements, including up to 120° rotation of the S4 segment, changes in hydrogen-bonding patterns, but only low amounts of translation. A smaller rotation (∼35°) of the extracellular end of all S4 segments is present also in a reference 0.5 µs simulation without applied field, which indicates that the crystal structure might be slightly different from the natural state of the voltage sensor. The conformation change upon hyperpolarization is closely coupled to an increase in 3₁₀ helix contents in S4, starting from the intracellular side. This could support a model for transition from the crystal structure where the hyperpolarization destabilizes S4–lipid hydrogen bonds, which leads to the helix rotating to keep the arginine side chains away from the hydrophobic phase, and the driving force for final relaxation by downward translation is partly entropic, which would explain the slow process. The coordinates of the transmembrane part of the simulated channel actually stay closer to the recently determined higher-resolution Kv1.2 chimera channel than the starting structure for the entire second half of the simulation (0.5–1 µs). Together with lipids binding in matching positions and significant thinning of the membrane also observed in experiments, this provides additional support for the predictive power of microsecond-scale membrane protein simulations.     

Conformational Studies of Nucleic Acids: IV. The Conformational Energetics of Oligonucleotides: d(ApApApA) and ApApApA

Abstract

Utilizing a new method for modeling furanose pseudorotation (D. A Pearlman and S.-H. Kim, J. Biomol. Struct. Dyn. 3, 85 (1985)) and the empirical multiple correlations between nucleic acid torsion angles we derived in the previous report (D. A Pearlman and S.-H. Kim, previous paper in this issue), we have made an energetic examination of the entire conformational spaces available to two nucleic acid oligonucleotides: d(ApApApA) and ApApApA The energies are calculated using a semi-empirical potential function. From the resulting body of data, energy contour map pairs (one for the DNA molecule, one for the RNA structure) have been created for each of the 21 possible torsion angle pairs in a nucleotide repeating unit. Of the 21 pairs, 15 have not been reported previously. The contour plots are different from those made earlier in that for each point in a particular angle-angle plot, the remaining five variable torsion angles are rotated to the values which give a minimum energy at this point. The contour maps are overall quite consistent with the experimental distribution of oligonucleotide data. A number of these maps are of particular interest: δ (C5′-C4′-C3′-03′)χ (04′-C1′-N9- C4), where the energetic basis for an approximately linear δ-χ correlation can be seen; ζ (C3′- 03′-P-05′)-δ, in which the experimentally observed linear correlation between ζ and δ in DNA (220° < ζ <280°) is clearly predicted; ζ-ε (C4′-C3′-03′-P), which shows that e increases with decreasing ζ <260°; α (03′-P-05′-C5′)-γ (05′-C5′-C4′-C3′) where a clear linear correlation between these angles is also apparent, consistent with experiment; and several others. For the DNA molecule studied here, the sugar torsion Ô is predicted to be the most flexible, while for the RNA molecule, the greatest amount of flexibility is expected to reside in a and y. Both the DNA and RNA molecules are predicted to be highly polymorphic. Complete energy minimization has been performed on each of the minima found in the energy searches and the results further support this prediction. Possible pathways for B-form to A-form DNA interconversion suggested by the results of this study are discussed. The results of these calculations support use of the new sugar modeling technique and torsion angle correlations in future conformational studies of nucleic acids.     

Systematics of the Osteocephalus buckleyi species complex (Anura,Hylidae) from Ecuador and Peru

We present a new phylogeny, based on DNA sequences of mitochondrial and nuclear genes, for frogs of the genus Osteocephalus with emphasis in the Osteocephalus buckleyi species complex. Genetic, morphologic, and advertisement call data are combined to define species boundaries and describe new species. The phylogeny shows strong support for: (1) a basal position of Osteocephalus taurinus + Osteocephalus oophagus, (2) a clade containing phytotelmata breeding species, and (3) a clade that corresponds to the Osteocephalus buckleyi species complex. Our results document a large proportion of hidden diversity within a set of populations that were previously treated as a single, widely distributed species, Osteocephalus buckleyi. Individuals assignable to Osteocephalus buckleyi formed a paraphyletic group relative to Osteocephalus verruciger and Osteocephalus cabrerai and contained four species, one of which is Osteocephalus buckleyi sensu stricto and three are new. Two of the new species are shared between Ecuador and Peru (Osteocephalus vilmae sp. n. and Osteocephalus cannatellai sp. n.) and one is distributed in the Amazon region of southern Peru (Osteocephalus germani sp. n.) We discuss the difficulties of using morphological characters to define species boundaries and propose a hypothesis to explain them.