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Vinesh Vijayan

Publications and source records attributed to Vinesh Vijayan.

8 recordsLinked to original sources
PubMed2005

Conkunitzin-S1 is the first member of a new Kunitz-type neurotoxin family. Structural and functional characterization.

Conkunitzin-S1 (Conk-S1) is a 60-residue neurotoxin from the venom of the cone snail Conus striatus that interacts with voltage-gated potassium channels. Conk-S1 shares sequence homology with Kunitz-type proteins but contains only two out of the three highly conserved cysteine bridges, which are typically found in these small, basic protein modules. In this study the three-dimensional structure of Conk-S1 has been solved by multidimensional NMR spectroscopy. The solution structure of recombinant Conk-S1 shows that a Kunitz fold is present, even though one of the highly conserved disulfide cross-links is missing. Introduction of a third, homologous disulfide bond into Conk-S1 results in a functional toxin with similar affinity for Shaker potassium channels. The affinity of Conk-S1 can be enhanced by a pore mutation within the Shaker channel pore indicating an interaction of Conk-S1 with the vestibule of potassium channels.

Amino Acid Sequence↗
PubMed2005

The nature of the stimulus and of the fumarate binding site of the fumarate sensor DcuS of Escherichia coli.

DcuS is a membrane-associated sensory histidine kinase of Escherichia coli specific for C(4) -dicarboxylates. The nature of the stimulus and its structural prerequisites were determined by measuring the induction of DcuS-dependent dcuB'-'lacZ gene expression. C(4)-dicarboxylates without or with substitutions at C2/C3 by hydrophilic (hydroxy, amino, or thiolate) groups stimulated gene expression in a similar way. When one carboxylate was replaced by sulfonate, methoxy, or nitro groups, only the latter (3-nitropropionate) was active. Thus, the ligand of DcuS has to carry two carboxylate or carboxylate/nitro groups 3.1-3.8 A apart from each other. The effector concentrations for half-maximal induction of dcuB'-'lacZ expression were 2-3 mm for the C(4)-dicarboxylates and 0.5 mm for 3-nitropropionate or d-tartrate. The periplasmic domain of DcuS contains a conserved cluster of positively charged or polar amino acid residues (Arg(107)-X(2)-His(110)-X(9)-Phe(120)-X(26)-Arg(147)-X-Phe(149)) that were essential for fumarate-dependent transcriptional regulation. The presence of fumarate or d-tartrate caused sharpening of peaks or chemical shift changes in HSQC NMR spectra of the isolated C(4)-dicarboylate binding domain. The amino acid residues responding to fumarate or d-tartrate were in the region comprising residues 89-150 and including the supposed binding site. DcuS(R147A) mutant with an inactivated binding site was isolated and reconstituted in liposomes. The protein showed the same (activation-independent) kinase activity as DcuS, but autophosphorylation of DcuS was no longer stimulated by C(4)-dicarboxylates. Therefore, the R147A mutation affected signal perception and transfer to the kinase but not the kinase activity per se.

Amino Acid Sequence↗
PubMed2005

Simultaneous measurement of protein one-bond residual dipolar couplings without increased resonance overlap.

A NMR strategy designed to measure simultaneously and without increased resonance overlap scalar and dipolar couplings (RDCs) in (13)C-, (15)N-labeled proteins is presented. Contrary to common schemes for simultaneous measurement of RDCs, a single reference experiment is used for the extraction of more than one type of coupling, thereby reducing the required measurement time. This is accomplished by a common reference spectrum followed by a series of interleaved experiments, in which a particular coupling dependent parameter is varied according to the quantitative J-correlation method or using accordion spectroscopy. To illustrate this idea, we have modified the 3D TROSY-HNCO and the 3D CBCA(CO)NH experiment allowing efficient measurement of one-bond (1)D(NH), (1)D(C'N), (1)D(CalphaHalpha), (1)D(CbetaHbeta), and (1)D(CalphaC') couplings in small to medium sized proteins. In addition, the experiments are expected to be useful for largely unfolded proteins, which show strong resonance overlap but have very favorable relaxation properties. Measurement of RDCs is demonstrated on uniformly (15)N-(13)C-labeled ubiquitin and on the sensory domain of the membraneous two-component fumarate sensor DcuS of Escherichia coli (17 kDa). DcuS was found to be unstable and to precipitate in one to two weeks. RDCs obtained from these experiments are in good agreement with the 1.8A X-ray structure of ubiquitin.

Carbon Isotopes↗
PubMed2004

Structure and DNA-binding properties of the cytolysin regulator CylR2 from Enterococcus faecalis.

Enterococcus faecalis is one of the major causes for hospital-acquired antibiotic-resistant infections. It produces an exotoxin, called cytolysin, which is lethal for a wide range of Gram-positive bacteria and is toxic to higher organisms. Recently, the regulation of the cytolysin operon was connected to autoinduction by a quorum-sensing mechanism involving the CylR1/CylR2 two-component regulatory system. We report here the crystal structure of CylR2 and its properties in solution as determined by heteronuclear NMR spectroscopy. The structure reveals a rigid dimer containing a helix-turn-helix DNA-binding motif as part of a five-helix bundle that is extended by an antiparallel beta-sheet. We show that CylR2 is a DNA-binding protein that binds specifically to a 22 bp fragment of the cytolysin promoter region. NMR chemical shift perturbation experiments identify surfaces involved in DNA binding and are in agreement with a model for the CylR2/DNA complex that attributes binding specificity to a complex network of CylR2/DNA interactions. Our results propose a mechanism where repression is achieved by CylR2 obstruction of the promoter preventing biosynthesis of the cytolysin operon transcript.

Bacterial Proteins↗
PubMed2003

The NMR structure of the sensory domain of the membranous two-component fumarate sensor (histidine protein kinase) DcuS of Escherichia coli.

The structure of the water-soluble, periplasmic domain of the fumarate sensor DcuS (DcuS-pd) has been determined by NMR spectroscopy in solution. DcuS is a prototype for a sensory histidine kinase with transmembrane signal transfer. DcuS belongs to the CitA family of sensors that are specific for sensing di- and tricarboxylates. The periplasmic domain is folded autonomously and shows helices at the N and the C terminus, suggesting direct linking or connection to helices in the two transmembrane regions. The structure constitutes a novel fold. The nearest structural neighbor is the Per-Arnt-Sim domain of the photoactive yellow protein that binds small molecules covalently. Residues Arg107, His110, and Arg147 are essential for fumarate sensing and are found clustered together. The structure constitutes the first periplasmic domain of a two component sensory system and is distinctly different from the aspartate sensory domain of the Tar chemotaxis sensor.

Amino Acid Sequence↗