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Mutations on EF-hands of potassium channel-interacting protein2.2 affect its interaction with Kv channel
No full textMutagenesis studies on the four EF-hands of KChIP2.2 (Potassium channel-interacting protein 2.2) were carried out to explore the conformational transition upon the binding of Ca2+ and Mg2+ and the subsequent effect on the interaction between KChIP2.2 and Kv4.2. CD spectra indicated that Ca2+- and Mg2+-loaded wild-type and mutated KChIP2.2 altered the secondary structure contents. In contrast to other mutants, mutation on EF1 caused a notably change in the secondary structure of KChIP2.2. Fluorescence measurement revealed that EF-hands 3 and 4 were high affinity Ca2+-binding sites within KChIP2.2 molecule, but the binding of Mg2+ with KChIP2.2 was marginally affected by EF-hand mutations. The results of size-exclusion chromatography showed that mutations on EF-1, EF-2 and EF-3 induced the oligomerization of KChIP2.2 and the extent of oligomerization was enhanced by Ca2+ and Mg2+. No significant differences were noted when wild-type and mutated KChIP2.2 bound with porcine brain membrane and liposome either in the absence or presence of Ca2+- and Mg2+. Pull down assay showed that KChIP2.2 and EF-hand mutants could bind with Kv4.2 in the absence of Ca2+ and Mg2+, but the interaction was enhanced by Ca2+ and Mg2+. However, the binding capability of mutants for Kv4.2 was notably lower than that observed for wild-type KChIP2.2. It was found that, in sharp contrast to that EF1 mutant exclusively localized in the nucleus, the other EF-hand mutants and wild-type protein distributed within nucleus as well as cytoplasm. Elevating intracellular Ca2+ concentration caused the translocation of EF1 mutant to cytoplasm but no appreciable effect on other mutants and wild-type KChIP2.2. . Taken together, these results suggest that the integrity of the four EF-hands are involved in function to stabilize conformation for binding with Kv channel, but this conformational transition is not essential for the binding to cell membrane
The structural elements of human visinin-like proteins functionally affect its conformational transition and regulate the activity of guanylyl cyclase
No full textIt has been well-known that VILIP-1 but not VILIP-3 regulates the activity of
guanylyl cyclase-B. In order to identify the modulated region within VILIP-1
on regulating guanylyl cyclase-B activity, the recombinant myristoylated and
nonmyristoylated VILIPs (VILIP-1, VILIP-3, chimeric VILIPs, and mutant
VILIP-1) were prepared in the present study. The recombinant proteins were
purified using ion-exchanger chromatography followed by gel filtration. CD
spectra indicated that the secondary structure of VILIPs was dominant with
\uce\ub1-helix, reflecting a well-conserved EF-hand structure. Tryptic digestion assay
and the fluorescence measurement showed that myristoylation, Ca2+ and Mg2+
differently induced the conformational changes of VILIPs. The results of gel
filtration chromatography reflected that the EF-3&4 of VILIP-1 and
myristoylation were involved in the dimerization of VILIP-1, and the dimer and
monomer were converted each other in a dynamic manner. The porcine brain
membrane binding assay and liposome binding assay showed that the binding
capability of VILIPs were markedly enhanced by myristoylation, Mg2+ and Ca2+.
Myristoylation and the intact EF-1 of VILIP-1 were found to essential for
the regulation of guanylyl cyclase activity in the presence of Mg2+ and Ca2+.
Taken together, theses results suggest that myristoylation and EF hand-1 of
VILIP-1 are the structural elements crucial for regulating the guanylyl cyclase
activity. In contrast to oligomerization of VILIP-1, Mg2+ and Ca2+ -induced
conformational changes of VILIP-1 and enhancement of the binding of VILIP-1
with membrane by Mg2+ and Ca2+ partly but not heavily involve in the action
Chemical modification of lysine residues and \uce\ub1-amino group affects the mechanism of Naja naja atra cardiotoxin 3 on damaging phospholipid vesicles
No full textTaiwan cobra (Naja naja atra) cardiotoxins, are composed of 60 amino acids and structurally stabilized by four disulfide bonds to form three loops structure. Cardiotoxins induced hemolysis of red blood cells and form a channel-like structure in inducing phospholipid vesicles leakage. The aim of the present study is to explore the events affect the membrane-damaging activity of cardiotoxin 3 (CTX3). Depletion of cholesterol in membrane of red blood cells using methyl-\uce\ub2-cyclodextrin (M\uce\ub2CD) enhanced the hemolytic activity of CTX3. Expectedly, CTX3 showed a low binding capability and lower membrane-damaging activity toward phospholipid vesicles containing cholesterol. Guanidinated of lysine residues and selective trinitrobenzoylation (TNP) at N-terminus of guanidinated CTX3 (Gu-CTX3) insignificantly altered binding capability and membrane-damaging activity of CTX3 regardless of phospholipid vesicles containing cholesterol. The binding of CTX3, Gu-CTX3 and TNP-Gu-CTX3 with phospholipid vesicles was reduced by increasing NaCl concentration. Consistent with these observations, membrane-damaging activity of CTX3, Gu-CTX3 and TNP-Gu-CTX3 were also decreased with increasing NaCl concentration. Unlike that of Gu-CTX3 and TNP-Gu-CTX3, membrane-damaging activity and membrane-induced oligomerization of CTX3 was completely abolished by 1 M NaCl. Fourier transform infrared spectroscopy indicated that CTX3, Gu-CTX3 and TNP-Gu-CTX3 adopted different conformation on binding with phospholipid vesicles and phospholipid/cholesterol vesicles. Taken together, our data indicate that membrane-bound conformation of CTX3 is affected by cholesterol and guanidination of lysine residues
Taiwan Banded Krait beta-Bungarotoxins: Novel Isotoxins, Targeting and Gene Organization
No full textbeta-Bungarotoxin (beta-Bgt), the presynaptic neurotoxin purified from the venom of Bungarus multicinctus, consists of the A chain and the B chain, cross-linked by an interchain disulfide bond. In this study, two novel beta-Bgt isotoxins were purified from Bungarus multicinctus venom by the combinations of ion-exchange chromatography and reverse phase HPLC. Amino acid sequencing, peptide mapping and mass analyses revealed that they probably contained the same A chain, but their B chain differed. Consequently, the discrepancies in their biological activity and fine structure reflected the role of B chain in intact of beta-Bgt. In Yeast-Two-Hybrid system, a potassium channel binding protein was identified to interact with the B chain of beta-Bgt. Although the recombinant potassium channel binding protein functionally bound with Ca2+, but it could not prove to bind with BM12 and BM13 as revealed by in vitro cross-linking assay.
The A chain genes including A1 chain, A2 chain and A8 chain genes were amplified by PCR reaction. Their nucleotide sequences shared up to 97.5% identity. Alignment of the determined A chain genes with A chain cDNAs revealed that the A1 chain gene was organized with four exon and three intron, while A2 chain gene comprised three exons and two introns. When A2 chain is expressed, the region corresponds to the first exon of A1 chain gene is skipped instead of inclusion of intronic sequence adjacent to the second intron. The resulting A2 chain mRNA encoded a 25 residues signal peptide, which different from A1 chain mRNA with a 27 residues signal peptide. Comparative analyses on phospholipase A2 genes and cDNAs suggest that this is the first report on skipping of exon changes the signal peptide sequence of snake venom proteins
Intermolecular interaction of KChIP proteins with beta-bungarotoxin and cardiotoxin
No full textAbstract
Our previous study showed that KChIP3 (Kv Channel Interacting Protein 3) probably was a physiological targete protein of beta-bungarotoxin (beta-Bgt) as evidenced by yeast two-hybrid system. Thus, extensive efforts are carried out to explore the molecular interaction between KChIP3 and beta-Bgt in the present study. KChIPs are potassium (Kv) channel-interacting proteins that bind to the 1~90 amino acid of N-terminus of Kv4 alpha-subunits and regulate the ion current density, shift the voltage dependence of activation and speed their recovery from inactivation. beta-Bgt, a presynaptic neurotoxin purified from Bungarus multicinctus venom, consists of A chain and B chain which cross-linked by an interchain disulfide bond. The results of pull-down assay revealed that, in contrast to other KChIP proteins, KChIP3 bound with beta-Bgt. Moreover, it was found that the B chain of beta-Bgt was a functional subunit in the binding with KChIP3, and the binding of KChIP3 to beta-Bgt showed a Ca2+-dependent manner. Removal of the third and the fourth EF-hand regions of KChIP3 abolished its interaction with beta-Bgt. Noticeably, the binding of beta-Bgt with KChIP3 did not influence the interaction between KChIP3 and Kv4. In the meantime, rat brain KChIP3 could be isolated using a beta-Bgt-Sepharose column. These observations suggest that KChIP3 is an intra-cellular target recognized by beta-Bgt. Accidently, it was found that direct protein-protein interaction between Taiwan cobra cardiotoxin3 (CTX3) and potassium channel-interacting proteins (KChIPs) was investigated. It was found that KChIPs bound with CTX3, in which KChIP1 and CTX3 formed a 1:1 complex as evidenced by the results of chemical crosslinking. Pull-down assay revealed that the intact EF-hand 3 and 4 of KChIP1 was critical for CTX3-binding. Whereas, all mutated KChIP3 were able to bind with CTX3. In contrast to the interaction between KChIP1 and KvN, the binding of CTX3 to KChIP1 showed a Ca2+-independent manner. Fluorescence measurement revealed that CTX3 affected the binding of ANS to Ca2+-bound KChIP1, but not Ca2+-free KChIP1. Alternatively, KChIP1 simultaneously bound with KvN and CTX3, and the interaction between KChIP1 and KvN was enhanced by CTX3. In terms of the fact that KChIPs regulate the electrophysiological properties of Kv K+ channel, the potentiality of beta-bgt and CTX for this biomedical application could be considered
The contribution of non-native structure with recombinant cobrotoxin to its immunoreactivity toward anti-cobrotoxin antibodies
No full textTo induce the production of antibodies, exogenous antigens are taken up and degraded in antigen presenting cells in vivo. Since this process inevitably lead to distort antigen\ue2s structure, it is likely that some arising antibodies following immunization may not react appropriately with native protein. In the present study, comparative studies on the reactivity of cobrotoxin and recombinant cobrotoxin toward anti-cobrotoxin antibodies were carried out. CD spectra and acrylamide quenching of Trp fluorescence showed that global structure of recombinant cobrotoxin was different from that of native toxin. Results of ELISA and dot blotting assay revealed that recombinant cobrotoxin had a superior reactivity toward anti-cobrotoxin antibodies than native toxin did. Reactivity with antibody fractions specifically against N-terminal region or C-terminal region of cobrotoxin also showed the same results. The binding of recombinant cobrotoxin with antibodies was stronger than that of cobrotoxin as revealed by ammonium thiocyanate elution assay. Recombinant protein was susceptible to reduce its antigenicity after tryptic digestion compared to cobrotoxin. Distorting disulfide linkages at C-terminus caused a marked decrease in immunoreactivity of recombinant cobrotoxin, indicating that anti-cobrotoxin antibodies mostly recognized conformation-dependent epitopes. Moreover, cobrotoxin and recombinant cobrotoxin showed a similar immunoreactivity under denaturing condition. Taken together, these results suggest that native conformation with cobrotoxin may unfavorably impede the interaction of some epitope(s) with anti-cobrotoxin antibodies
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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