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Structural Determinants (structural + determinant)
Selected AbstractsSYMPOSIUM REVIEW: Revealing the structural basis of action of hERG potassium channel activators and blockersTHE JOURNAL OF PHYSIOLOGY, Issue 17 2010Matthew Perry Human ether-á-go-go related gene (hERG) potassium (K+) channels play a critical role in cardiac action potential repolarization. This is due, in large part, to the unique gating properties of these channels, which are characterized by relatively slow activation and an unusually fast and voltage-dependent inactivation. A large number of structurally diverse compounds bind to hERG and carry an unacceptably high risk of causing arrhythmias. On the other hand, drugs that increase hERG current may, at least in principle, prove useful for treatment of long QT syndrome. A few blockers have been shown to increase hERG current at potentials close to the threshold for channel activation , a process referred to as facilitation. More recently, a novel group of hERG channel activators have been identified that slow deactivation and/or attenuate inactivation. Structural determinants for the action of two different types of activators have been identified. These compounds bind at sites that are distinct from each other and also separate from the binding site of high affinity blockers. They reveal not only novel ways of chemically manipulating hERG channel function, but also interactions between structural domains that are critical to normal activation and inactivation gating. [source] Identification of the structural determinant responsible for the phosphorylation of G-protein activated potassium channel 1 by cAMP-dependent protein kinaseFEBS JOURNAL, Issue 21 2009Carmen Müllner Besides being activated by G-protein ,/, subunits, G-protein activated potassium channels (GIRKs) are regulated by cAMP-dependent protein kinase. Back-phosphorylation experiments have revealed that the GIRK1 subunit is phosphorylated in vivo upon protein kinase A activation in Xenopus oocytes, whereas phosphorylation was eliminated when protein kinase A was blocked. In vitro phosphorylation experiments using truncated versions of GIRK1 revealed that the structural determinant is located within the distant, unique cytosolic C-terminus of GIRK1. Serine 385, serine 401 and threonine 407 were identified to be responsible for the incorporation of radioactive 32P into the protein. Furthermore, the functional effects of cAMP injections into oocytes on currents produced by GIRK1 homooligomers were significantly reduced when these three amino acids were mutated. The data obtained in the present study provide information about the structural determinants that are responsible for protein kinase A phosphorylation and the regulation of GIRK channels. Structured digital abstract ,,MINT-7260296, MINT-7260317, MINT-7260333, MINT-7260347, MINT-7260361, MINT-7260270: PKA-cs (uniprotkb:P00517) phosphorylates (MI:0217) Girk1 (uniprotkb:P63251) by protein kinase assay (MI:0424) [source] Activation of phospholipase C pathways by a synthetic chondroitin sulfate-E tetrasaccharide promotes neurite outgrowth of dopaminergic neuronsJOURNAL OF NEUROCHEMISTRY, Issue 2 2007Naoki Sotogaku Abstract In dopaminergic neurons, chondroitin sulfate (CS) proteoglycans play important roles in neuronal development and regeneration. However, due to the complexity and heterogeneity of CS, the precise structure of CS with biological activity and the molecular mechanisms underlying its influence on dopaminergic neurons are poorly understood. In this study, we investigated the ability of synthetic CS oligosaccharides and natural polysaccharides to promote the neurite outgrowth of mesencephalic dopaminergic neurons and the signaling pathways activated by CS. CS-E polysaccharide, but not CS-A, -C or -D polysaccharide, facilitated the neurite outgrowth of dopaminergic neurons at CS concentrations within the physiological range. The stimulatory effect of CS-E polysaccharide on neurite outgrowth was completely abolished by its digestion into disaccharide units with chondroitinase ABC. Similarly to CS-E polysaccharide, a synthetic tetrasaccharide displaying only the CS-E sulfation motif stimulated the neurite outgrowth of dopaminergic neurons, whereas a CS-E disaccharide or unsulfated tetrasaccharide had no effect. Analysis of the molecular mechanisms revealed that the action of the CS-E tetrasaccharide was mediated through midkine-pleiotrophin/protein tyrosine phosphatase , and brain-derived neurotrophic factor/tyrosine kinase B receptor pathways, followed by activation of the two intracellular phospholipase C (PLC) signaling cascades: PLC/protein kinase C and PLC/inositol 1,4,5-triphosphate/inositol 1,4,5-triphosphate receptor signaling leading to intracellular Ca2+ concentration-dependent activation of Ca2+/calmodulin-dependent kinase II and calcineurin. These results indicate that a specific sulfation motif, in particular the CS-E tetrasaccharide unit, represents a key structural determinant for activation of midkine, pleiotrophin and brain-derived neurotrophic factor-mediated signaling, and is required for the neuritogenic activity of CS in dopaminergic neurons. [source] Identification of the structural determinant responsible for the phosphorylation of G-protein activated potassium channel 1 by cAMP-dependent protein kinaseFEBS JOURNAL, Issue 21 2009Carmen Müllner Besides being activated by G-protein ,/, subunits, G-protein activated potassium channels (GIRKs) are regulated by cAMP-dependent protein kinase. Back-phosphorylation experiments have revealed that the GIRK1 subunit is phosphorylated in vivo upon protein kinase A activation in Xenopus oocytes, whereas phosphorylation was eliminated when protein kinase A was blocked. In vitro phosphorylation experiments using truncated versions of GIRK1 revealed that the structural determinant is located within the distant, unique cytosolic C-terminus of GIRK1. Serine 385, serine 401 and threonine 407 were identified to be responsible for the incorporation of radioactive 32P into the protein. Furthermore, the functional effects of cAMP injections into oocytes on currents produced by GIRK1 homooligomers were significantly reduced when these three amino acids were mutated. The data obtained in the present study provide information about the structural determinants that are responsible for protein kinase A phosphorylation and the regulation of GIRK channels. Structured digital abstract ,,MINT-7260296, MINT-7260317, MINT-7260333, MINT-7260347, MINT-7260361, MINT-7260270: PKA-cs (uniprotkb:P00517) phosphorylates (MI:0217) Girk1 (uniprotkb:P63251) by protein kinase assay (MI:0424) [source] Site-directed mutagenesis of selected residues at the active site of aryl-alcohol oxidase, an H2O2 -producing ligninolytic enzymeFEBS JOURNAL, Issue 21 2006Patricia Ferreira Aryl-alcohol oxidase provides H2O2 for lignin biodegradation, a key process for carbon recycling in land ecosystems that is also of great biotechnological interest. However, little is known of the structural determinants of the catalytic activity of this fungal flavoenzyme, which oxidizes a variety of polyunsaturated alcohols. Different alcohol substrates were docked on the aryl-alcohol oxidase molecular structure, and six amino acid residues surrounding the putative substrate-binding site were chosen for site-directed mutagenesis modification. Several Pleurotus eryngii aryl-alcohol oxidase variants were purified to homogeneity after heterologous expression in Emericella nidulans, and characterized in terms of their steady-state kinetic properties. Two histidine residues (His502 and His546) are strictly required for aryl-alcohol oxidase catalysis, as shown by the lack of activity of different variants. This fact, together with their location near the isoalloxazine ring of FAD, suggested a contribution to catalysis by alcohol activation, enabling its oxidation by flavin-adenine dinucleotide (FAD). The presence of two aromatic residues (at positions 92 and 501) is also required, as shown by the conserved activity of the Y92F and F501Y enzyme variants and the strongly impaired activity of Y92A and F501A. By contrast, a third aromatic residue (Tyr78) does not seem to be involved in catalysis. The kinetic and spectral properties of the Phe501 variants suggested that this residue could affect the FAD environment, modulating the catalytic rate of the enzyme. Finaly, L315 affects the enzyme kcat, although it is not located in the near vicinity of the cofactor. The present study provides the first evidence for the role of aryl-alcohol oxidase active site residues. [source] Loss-of-function variants of the human melanocortin-1 receptor gene in melanoma cells define structural determinants of receptor functionFEBS JOURNAL, Issue 24 2002Jesús Sánchez Más The ,-melanocyte-stimulating hormone (,MSH) receptor (MC1R) is a major determinant of mammalian skin and hair pigmentation. Binding of ,MSH to MC1R in human melanocytes stimulates cell proliferation and synthesis of photoprotective eumelanin pigments. Certain MC1R alleles have been associated with increased risk of melanoma. This can be theoretically considered on two grounds. First, gain-of-function mutations may stimulate proliferation, thus promoting dysplastic lesions. Second, and opposite, loss-of-function mutations may decrease eumelanin contents, and impair protection against the carcinogenic effects of UV light, thus predisposing to skin cancers. To test these possibilities, we sequenced the MC1R gene from seven human melanoma cell (HMC) lines and three giant congenital nevus cell (GCNC) cultures. Four HMC lines and two GCNC cultures contained MC1R allelic variants. These were the known loss-of-function Arg142His and Arg151Cys alleles and a new variant, Leu93Arg. Moreover, impaired response to a superpotent ,MSH analog was demonstrated for the cell line carrying the Leu93Arg allele and for a HMC line homozygous for wild-type MC1R. Functional analysis in heterologous cells stably or transiently expressing this variant demonstrated that Leu93Arg is a loss-of-function mutation abolishing agonist binding. These results, together with site-directed mutagenesis of the vicinal Glu94, demonstrate that the MC1R second transmembrane fragment is critical for agonist binding and maintenance of a resting conformation, whereas the second intracellular loop is essential for coupling to the cAMP system. Therefore, loss-of-function, but not activating MC1R mutations are common in HMC. Their study provides important clues to understand MC1R structure-function relationships. [source] Soluble oligomers from a non-disease related protein mimic A,-induced tau hyperphosphorylation and neurodegenerationJOURNAL OF NEUROCHEMISTRY, Issue 2 2007Marcelo N. N. Vieira Abstract Protein aggregation and amyloid accumulation in different tissues are associated with cellular dysfunction and toxicity in important human pathologies, including Alzheimer's disease and various forms of systemic amyloidosis. Soluble oligomers formed at the early stages of protein aggregation have been increasingly recognized as the main toxic species in amyloid diseases. To gain insight into the mechanisms of toxicity instigated by soluble protein oligomers, we have investigated the aggregation of hen egg white lysozyme (HEWL), a normally harmless protein. HEWL initially aggregates into ,-sheet rich, roughly spherical oligomers which appear to convert with time into protofibrils and mature amyloid fibrils. HEWL oligomers are potently neurotoxic to rat cortical neurons in culture, while mature amyloid fibrils are little or non-toxic. Interestingly, when added to cortical neuronal cultures HEWL oligomers induce tau hyperphosphorylation at epitopes that are characteristically phosphorylated in neurons exposed to soluble oligomers of the amyloid-, peptide. Furthermore, injection of HEWL oligomers in the cerebral cortices of adult rats induces extensive neurodegeneration in different brain areas. These results show that soluble oligomers from a non-disease related protein can mimic specific neuronal pathologies thought to be induced by soluble amyloid-, peptide oligomers in Alzheimer's disease and support the notion that amyloid oligomers from different proteins may share common structural determinants that would explain their generic cytotoxicities. [source] DE-loop mutations affect ,2 microglobulin stability, oligomerization, and the low-pH unfolded formPROTEIN SCIENCE, Issue 7 2010Carlo Santambrogio Abstract ,2 microglobulin (,2m) is the light chain of class-I major histocompatibility complex (MHC-I). Its accumulation in the blood of patients affected by kidney failure leads to amyloid deposition around skeletal joints and bones, a severe condition known as Dialysis Related Amyloidosis (DRA). In an effort to dissect the structural determinants of ,2m aggregation, several ,2m mutants have been previously studied. Among these, three single-residue mutations in the loop connecting strands D and E (W60G, W60V, D59P) have been shown to affect ,2m amyloidogenic properties, and are here considered. To investigate the biochemical and biophysical properties of wild-type (w.t.) ,2m and the three mutants, we explored thermal unfolding by Trp fluorescence and circular dichroism (CD). The W60G mutant reveals a pronounced increase in conformational stability. Protein oligomerization and reduction kinetics were investigated by electrospray-ionization mass spectrometry (ESI-MS). All the mutations analyzed here reduce the protein propensity to form soluble oligomers, suggesting a role for the DE-loop in intermolecular interactions. A partially folded intermediate, which may be involved in protein aggregation induced by acids, accumulates for all the tested proteins at pH 2.5 under oxidizing conditions. Moreover, the kinetics of disulfide reduction reveals specific differences among the tested mutants. Thus, ,2m DE-loop mutations display long-range effects, affecting stability and structural properties of the native protein and its low-pH intermediate. The evidence presented here hints to a crucial role played by the DE-loop in determining the overall properties of native and partially folded ,2m. [source] The hemoglobins of the trematodes Fasciola hepatica and Paramphistomum epiclitum: A molecular biological, physico-chemical, kinetic, and vaccination studyPROTEIN SCIENCE, Issue 10 2008Sylvia Dewilde Abstract The trematode Fasciola hepatica (Fa.he.) is a common parasite of human and livestock. The hemoglobin (Hb) of Fa.he., a potential immunogen, was chosen for characterization in the search for an effective vaccine. Characterization of trematode Hbs show that they are intracellular single-domain globins with the following remarkable features: (1) Fa.he. expresses two Hb isoforms that differ at two amino acid sites (F1: 119Y/123Q; F2: 119F/123L). Both isoforms are monoacetylated at their N-termini; (2) the genes coding for Fa.he. and Paramphistomum epiclitum (Pa.ep.) Hbs are interrupted by two introns at the conserved positions B12.2 and G7.0.; (3) UV/VIS and resonance Raman spectroscopy identify the recombinant Fa.he. HbF2 as a pentacoordinated high-spin ferrous Hb; (4) electron paramagnetic resonance spectroscopy of cyano-met Fa.he. HbF2 proves that the endogenously bound imidazole has no imidazolate character; (5) the major structural determinants of the globin fold are present, they contain a TyrB10/TyrE7 residue pair on the distal side. Although such distal-site pair is a signature for high oxygen affinity, as shown for Pa.ep. Hb, the oxygen-binding rate parameters for Fa.he. Hb are intermediate between those of myoglobin and those of other trematode Hbs; (6) the three-dimensional structure of recombinant Fa.he. HbF2 from this study closely resembles the three-dimensional structure of Pa.ep. determined earlier. The set of distal-site polar interactions observed in Pa.ep. Hb is matched with small but significant structural adjustments; (7) despite the potential immunogenic character of the fluke Hb, vaccination of calves with recombinant Fa.he. HbF2 failed to promote protection against parasitic infection. [source] Molecular determinants of inactivation in voltage-gated Ca2+ channelsTHE JOURNAL OF PHYSIOLOGY, Issue 2 2000Steffen Hering Evolution has created a large family of different classes of voltage-gated Ca2+ channels and a variety of additional splice variants with different inactivation properties. Inactivation controls the amount of Ca2+ entry during an action potential and is, therefore, believed to play an important role in tissue-specific Ca2+ signalling. Furthermore, mutations in a neuronal Ca2+ channel (Cav2.1) that are associated with the aetiology of neurological disorders such as familial hemiplegic migraine and ataxia cause significant changes in the process of channel inactivation. Ca2+ channels of a given subtype may inactivate by three different conformational changes: a fast and a slow voltage-dependent inactivation process and in some channel types by an additional Ca2+ -dependent inactivation mechanism. Inactivation kinetics of Ca2+ channels are determined by the intrinsic properties of their pore-forming ,1 -subunits and by interactions with other channel subunits. This review focuses on structural determinants of Ca2+ channel inactivation in different parts of Ca2+ channel ,1 -subunits, including pore-forming transmembrane segments and loops, intracellular domain linkers and the carboxyl terminus. Inactivation is also affected by the interaction of the ,1 -subunits with auxiliary ,-subunits and intracellular regulator proteins. The evidence shows that pore-forming S6 segments and conformational changes in extra- (pore loop) and intracellular linkers connected to pore-forming segments may play a principal role in the modulation of Ca2+ channel inactivation. Structural concepts of Ca2+ channel inactivation are discussed. [source] Can human rights discourse improve the health of Indigenous Australians?AUSTRALIAN AND NEW ZEALAND JOURNAL OF PUBLIC HEALTH, Issue 5 2006Natalie Gray Objective:Recognition of the poor health outcomes of Indigenous Australians has led to an interest in using human rights discourse as a framework for arguing that the Australian Government has an international obligation to improve Indigenous health. Method:This paper explores two potential directions for human rights discourse in this context. The first is the development and elaboration of an asserted ,human right to health'. The second focuses on developing an understanding of the interactions between health and human rights, particularly the underlying social determinants of health, and thereby creating an advocacy framework that could be used to promote the inclusion of human rights considerations into the policy-making agenda. Results:This paper argues that despite the symbolic force of human rights discourse, its capacity to improve the health of Indigenous Australians through international law is limited. This is so irrespective of whether recourse is made to a legal or moral imperative. Conclusion and Implications:The ,human right to health' is limited primarily by several barriers to its implementation, some of which are perpetuated by the current Australian Government itself. Although the potential advocacy capacity of human rights discourse is similarly limited by the hostility of the Government towards the notion of incorporating human rights considerations into its public policy decision making, it does provide a sustainable intellectual framework in which to consider the social and structural determinants of health and maintain these issues on the political agenda. [source] Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of the catalytic domain of a hyperthermostable endo-1,4-,- d -mannanase from Thermotoga petrophila RKU-1ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010Camila Ramos Santos Endo-1,4-,- d -mannanases play key roles in seed germination and fruit ripening and have recently received much attention owing to their potential applications in the food, detergent and kraft pulp industries. In order to delineate their structural determinants for specificity and stability, X-ray crystallographic investigations combined with detailed functional studies are being performed. In this work, crystals of the catalytic domain of a hyperthermostable endo-1,4-,- d -mannanase from Thermotoga petrophila RKU-1 were obtained from three different conditions, resulting in two crystalline forms. Crystals from conditions with phosphate or citrate salts as precipitant (CryP) belonged to space group P212121, with unit-cell parameters a = 58.76, b = 87.99, c = 97.34,Å, while a crystal from a condition with ethanol as precipitant (CryE) belonged to space group I212121, with unit-cell parameters a = 91.03, b = 89.97, c = 97.89,Å. CryP and CryE diffracted to resolutions of 1.40 and 1.45,Å, respectively. [source] Crystallization and preliminary X-ray analysis of the chemokine-binding protein from orf virus (Poxviridae)ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2010Rafael Miguez Couñago The parapoxvirus orf virus (ORFV) encodes a chemokine-binding protein (CBP) that functions to downregulate the host's immune response at the site of infection by blocking the chemokine-induced recruitment of immune cells. In order to shed light on the structural determinants of CBP,chemokine binding, ORFV CBP was crystallized as part of an ongoing structure,function study on this protein. ORFV CBP crystals were obtained by the sitting-drop vapour-diffusion technique using ammonium citrate as a precipitant. The crystal quality was greatly improved through the addition of small-molecule additives to the crystallization mother liquor. ORFV CBP crystals diffracted X-rays to 2.50,Å resolution and belonged to the hexagonal space group P6122 or its enantiomorph P6522, with unit-cell parameters a = b = 75.62, c = 282.49,Å, , = 90, , = 90, , = 120°. [source] Production, purification and preliminary X-ray crystallographic studies of adeno-associated virus serotype 9ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2009Matthew Mitchell Adeno-associated virus (AAV) serotype 9, which is under development for gene-delivery applications, shows significantly enhanced capsid-associated transduction efficiency in muscle compared with other AAV serotypes. With the aim of characterizing the structural determinants of this property, the purification, crystallization and preliminary X-ray crystallographic analyses of the AAV9 viral capsid are reported. The crystals diffracted X-rays to 2.8,Å resolution using synchrotron radiation and belonged to the trigonal space group P32, with unit-cell parameters a = b = 251.0, c = 640.0,Å. There are three complete viral capsids in the crystal unit cell. The orientation and position of the asymmetric unit capsid have been determined by molecular-replacement methods and structure determination is in progress. [source] Crystallization and crystallographic analysis of Bacillus subtilis xylanase CACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009Franz J. St John The recent biochemical characterization of the xylanases of glycosyl hydrolase family 5 (GH 5) has identified a distinctive endo mode of action, hydrolyzing the ,-1,4 xylan chain at a specific site directed by the position of an ,-1,2-linked glucuronate moiety. Xylanase C (XynC), the GH 5 xylanase from Bacillus subtilis 168, has been cloned, overexpressed and crystallized. Initial data collection was performed and a preliminary model has been built into a low-quality 2.7,Å resolution density map. The crystals belonged to the primitive monoclinic space group P21. Further screening identified an additive that resulted in large reproducible crystals. This larger more robust crystal form belonged to space group P21212 and a resulting data set has been processed to 1.64,Å resolution. This will be the second structure to be solved from this unique xylanase family and the first from a Gram-positive bacterium. This work may help to identify the structural determinants that allow the exceptional specificity of this enzyme and the role it plays in the biological depolymerization and processing of glucuronoxylan. [source] Probing the active site of MIO-dependent aminomutases, key catalysts in the biosynthesis of ,-amino acids incorporated in secondary metabolitesBIOPOLYMERS, Issue 9 2010Heather A. Cooke Abstract The tyrosine aminomutase SgTAM produces (S)-ß-tyrosine from L -tyrosine in the biosynthesis of the enediyne antitumor antibiotic C-1027. This conversion is promoted by the methylideneimidazole-5-one (MIO) prosthetic group. MIO was first identified in the homologous family of ammonia lyases, which deaminate aromatic amino acids to form ,,ß-unsaturated carboxylates. Studies of substrate specificity have been described for lyases but there have been limited reports in altering the substrate specificity of aminomutases. Furthermore, it remains unclear as to what structural properties are responsible for catalyzing the presumed readdition of the amino group into the ,,ß-unsaturated intermediates to form ß-amino acids. Attempts to elucidate specificity and mechanistic determinants of SgTAM have also proved to be difficult as it is recalcitrant to perturbations to the active site via mutagenesis. An X-ray cocrystal structure of the SgTAM mutant of the catalytic base with L -tyrosine verified important substrate binding residues as well as the enzymatic base. Further mutagenesis revealed that removal of these crucial interactions renders the enzyme inactive. Proposed structural determinants for mutase activity probed via mutagenesis, time-point assays and X-ray crystallography revealed a complicated role for these residues in maintaining key quaternary structure properties that aid in catalysis. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 802,810, 2010. [source] Computational analysis of glycoside hydrolase family 1 specificitiesBIOPOLYMERS, Issue 11 2008Anthony D. Hill Abstract Glycoside hydrolase family 1 consists of ,-glucosidases, ,-galactosidases, 6-phospho-,-galactosidases, myrosinases, and other enzymes having similar primary and tertiary structures but diverse specificities. Among these enzymes, ,-glucosidases hydrolyze cellobiose to glucose, and therefore they are key players in any cellulose to glucose process. All family members attack ,-glycosidic bonds between a pyranosyl glycon and an aglycon, but most have little specificity for the aglycon or for the bond configuration. Furthermore, glycon specificity is not absolute. Sixteen family members (six ,-glucosidases, two cyanogenic ,-glucosidases, one 6-phospho-,-galactosidase, two myrosinases, and five ,-glycosidases) have known tertiary structures. We have used automated docking to computationally bind disaccharides with allopyranosyl, galactopyranosyl, glucopyranosyl, mannopyranosyl, 6-phosphogalactopyranosyl, and 6-phosphoglucopyranosyl glycons, all linked by ,-(1,2), ,-(1,3), ,-(1,4), and ,-(1,6)-glycosidic bonds to ,-glucopyranoside aglycons, along with ,-(1,1-thio)-allopyranosyl, -galactopyranosyl, -glucopyranosyl, and -mannopyranosyl) ,-glucopyranosides, into all of these structures to investigate the structural determinants of their enzyme specificities. The following are the eight active-site residues: Glu191, Thr194, Phe205, Asn285, Arg336, Asn376, Trp378, and Trp465 (Zea mays ,-glucosidase numbering), that control a significant amount of glycon specificity. © 2008 Wiley Periodicals, Inc. Biopolymers 89: 1021,1031, 2008. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Solution structure of the cyclic peptide contryphan-Vn, a Ca2+ -dependent K+ channel modulatorBIOPOLYMERS, Issue 3 2004Tommaso Eliseo Abstract The solution structure of contryphan-Vn, a cyclic peptide with a double cysteine S,S bridge and containing a D -tryptophan extracted from the venom of the cone snail Conus ventricosus, has been determined by NMR spectroscopy using a variety of homonuclear and heteronuclear NMR methods and restrained molecular dynamics simulations. The main conformational features of backbone contryphan-Vn are a type IV ,-turn from Gly 1 to Lys 6 and a type I ,-turn from Lys 6 to Cys 9. As already found in other contryphans, one of the two prolines,the Pro4,is mainly in the cis conformation while Pro7 is trans. A small hydrophobic region probably partly shielded from solvent constituted from the close proximity of side chains of Pro7 and Trp8 was observed together with a persistent salt bridge between Asp2 and Lys6, which has been revealed by the diagnostic observation of specific nuclear Overhauser effects. The salt bridge was used as a restraint in the molecular dynamics in vacuum but without inserting explicit electrostatic contribution in the calculations. The backbone of the unique conformational family found of contryphan-Vn superimposes well with those of contryphan-Sm and contryphan-R. This result indicates that the contryphan structural motif represents a robust and conserved molecular scaffold whose main structural determinants are the size of the intercysteine loop and the presence and location in the sequence of the D -Trp and the two Pro residues. © 2004 Wiley Periodicals, Inc. Biopolymers, 2004 [source] Biological activities of Bv8 analoguesBRITISH JOURNAL OF PHARMACOLOGY, Issue 5 2005Lucia Negri The small protein Bv8, secreted by the skin of the frog Bombina variegata, belongs to a novel family of secreted proteins whose orthologues have been identified in snakes (MIT) and in mammals (prokineticins (PKs)). A characteristic feature of this protein family is the same N-terminal sequence, AVITGA, and the presence of 10 cysteines with identical spacing in the C-terminal domain. Two closely related G protein-coupled receptors that mediate signal transduction of Bv8/PKs have been cloned (PK-R1 and PK-R2). In mammals, the Bv8/PK protein family is involved in a number of biological activities such as ingestive behaviours, circadian rhythms, angiogenesis and pain sensitization. In an attempt to identify the structural determinants required for the pronociceptive activity of Bv8, we prepared Bv8 derivatives lacking one (des-Ala- Bv8) or two (des-Ala-Val -Bv8) residues from the N-terminus. des-Ala- Bv8 displayed a receptor affinity five times lower than that of Bv8, it was five times less potent in inducing [Ca2+]i transients and in causing p42/p44 MAPK phosphorylation in CHO-cells expressing PK-R1 and PK-R2. Moreover, dA-Bv8 was about 20 times less potent than Bv8 in inducing hyperalgesia in rats. The deletion of the first two amino acids of Bv8 abolished any biological activity both ,in vitro' and ,in vivo'; however, des-AlaVal -Bv8 is able to antagonize the Bv8-induced hyperalgesia, binding the PK-Rs on peripheral and central projections of the primary sensitive neurons. British Journal of Pharmacology (2005) 146, 625,632. doi:10.1038/sj.bjp.0706376 [source] Primary Steps of pH-Dependent Insulin Aggregation Kinetics are Governed by Conformational FlexibilityCHEMBIOCHEM, Issue 11 2009Jürgen Haas Dr. Abstract Insulin aggregation critically depends on pH. The underlying energetic and structural determinants are, however, unknown. Here, we measure the kinetics of the primary aggregation steps of the insulin monomer in vitro and relate it to its conformational flexibility. To assess these primary steps the monomer concentration was monitored by mass spectrometry at various pH values and aggregation products were imaged by atomic force microscopy. Lowering the pH from 3 to 1.6 markedly accelerated the observed aggregation kinetics. The influence of pH on the monomer structure and dynamics in solution was studied by molecular dynamics simulations, with the protonation states of the titrable groups obtained from electrostatic calculations. Reduced flexibility was observed for low pH values, mainly in the C terminus and in the helix of the B chain; these corresponded to an estimated entropy loss of 150 J,mol,1,K,1. The striking correlation between entropy loss and pH value is consistent with the observed kinetic traces. In analogy to the well-known , value analysis, this result allows the extraction of structural information about the rate determining transition state of the primary aggregation steps. In particular, we suggest that the residues in the helix of the B chain are involved in this transition state. [source] High-Throughput Substrate Specificity Studies of Sialidases by Using Chemoenzymatically Synthesized Sialoside LibrariesCHEMBIOCHEM, Issue 2 2007Harshal A. Chokhawala Abstract Sialidases, or neuraminidases, are enzymes that cleave terminal sialic acid (Sia) residues from complex sialic acid-containing structures. They have been found in many animals and microorganisms and are important in various physiological and pathological processes. In order to understand the biological significance of diverse sialidases, it is important to study in detail the structural determinants of their natural substrates. Here, we report the synthesis of sialoside libraries containing para -nitrophenol-tagged sialosides with different naturally occurring sialic acid forms, different sialyl linkages, and different penultimate monosaccharides using a highly efficient one-pot three-enzyme chemoenzymatic approach. By using these compounds in a 96-well plate-based colorimetric high-throughput screening platform, the diversity of substrate preference is shown for seven bacterial sialidases. The sialoside libraries and the screening method are convenient tools for unravelling the substrate specificity and the biological function of sialidases. [source] The Conformations of Amino Acids on a Gold(111) SurfaceCHEMPHYSCHEM, Issue 8 2010Martin Hoefling Abstract The interactions of amino acids with inorganic surfaces are of interest for biologists and biotechnologists alike. However, the structural determinants of peptide,surface interactions have remained elusive, but are important for a structural understanding of the interactions of biomolecules with gold surfaces. Molecular dynamics simulations are a tool to analyze structures of amino acids on surfaces. However, such an approach is challenging due to lacking parameterization for many surfaces and the polarizability of metal surfaces. Herein, we report DFT calculations of amino acid fragments in vacuo and molecular dynamics simulations of the interaction of all amino acids with a gold(111) surface in explicit solvent, using the recently introduced polarizable gold force field GolP. We describe preferred orientations of the amino acids on the metal surface. We find that all amino acids preferably interact with the gold surface at least partially with their backbone, underlining an unfolding propensity of gold surfaces. [source] | ||||||||||||||||