Dot Far-Western Blot Analysis of Relative Binding Affinities of the Src Homology 3 Domains of Efs and Its Related Proteins

Ohba, Takeaki; Ishino, Masaho; Aoto, Hiroshi; Sasaki, Terukatsu

doi:10.1006/abio.1998.2772

Cited by 14 publications

(6 citation statements)

References 28 publications

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“…The dot-far Western blotting method of Ohba et al (30) was performed with the following modifications. Aliquots containing 3 and 6 pmol of purified MglA-His fusion protein (or BSA for controls) were absorbed to nitrocellulose.…”

Section: Methodsmentioning

confidence: 99%

AglZ Is a Filament-Forming Coiled-Coil Protein Required for Adventurous Gliding Motility ofMyxococcus xanthus

et al. 2004

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The aglZ gene of Myxococcus xanthus was identified from a yeast two-hybrid assay in which MglA was used as bait. MglA is a 22-kDa cytoplasmic GTPase required for both adventurous and social gliding motility and sporulation. Genetic studies showed that aglZ is part of the A motility system, because disruption or deletion of aglZ abolished movement of isolated cells and aglZ sglK double mutants were nonmotile. The aglZ gene encodes a 153-kDa protein that interacts with purified MglA in vitro. The N terminus of AglZ shows similarity to the receiver domain of two-component response regulator proteins, while the C terminus contains heptad repeats characteristic of coiled-coil proteins, such as myosin. Consistent with this motif, expression of AglZ in Escherichia coli resulted in production of striated lattice structures. Similar to the myosin heavy chain, the purified C-terminal coiled-coil domain of AglZ forms filament structures in vitro.

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Section: Methodsmentioning

confidence: 99%

AglZ Is a Filament-Forming Coiled-Coil Protein Required for Adventurous Gliding Motility ofMyxococcus xanthus

et al. 2004

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“…The Cas proteins have a conserved domain structure composed of an amino terminal SH3 domain, a substrate domain which contains multiple tyrosine motifs that are recognized by SH2 domain proteins following phosphorylation, a serine rich region, and a carboxyl terminal dimerization motif (51). HEF1, pl30Cas, and Efs localize to focal adhesion sites via interaction of their SH3 domains with FAK (53,(60)(61)(62), and contribute to the assembly of signaling complexes downstream of the integrin receptor following ligand binding (51). An important question is whether the function of discrete Cas family members at focal complexes is equivalent, or whether individual Cas proteins are associated with promotion of different biological effects.…”

Section: Discussionmentioning

confidence: 99%

FCCC Institutional Breast Cancer Training Program

Russo¹

2001

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“…To evaluate the affinity and specificity of binding of rN domain to rC-domain, Far-dot ELISA technique was preformed according to Ohba et al [19] with minor modifications. Briefly, recombinant C-domain was spotted onto Nitrocellulose membrane and the membrane was treated with 5% skim milk solution for 1 h to saturate and block all the free binding sites.…”

Section: Methodsmentioning

confidence: 99%

In Silico, In Vitro and In Vivo Analysis of Binding Affinity between N and C-Domains of Clostridium perfringens Alpha Toxin

et al. 2013

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Clostridium perfringens alpha toxin/phospholipase C (CP-PLC) is one of the most potent bacterial toxins known to cause soft tissue infections like gas gangrene in humans and animals. It is the first bacterial toxin demonstrated to be an enzyme with phospholipase, sphingomyelinase and lecithinase activities. The toxin is comprised of an enzymatic N-domain and a binding C-domain interconnected by a flexible linker. The N-domain alone is non-toxic to mammalian cells, but incubation with C-domain restores the toxicity, the mechanism of which is still not elucidated. The objectives of the current study were to investigate the formation of a stable N and C-domain complex, to determine possible interactions between the two domains in silico and to characterize the in vitro and in vivo correlates of the interaction. To establish the existence of a stable N and C-domain hybrid, in vitro pull down assay and dot-Far Western blotting assays were employed, where it was clearly revealed that the two domains bound to each other to form an intermediate. Using bioinformatics tools like MetaPPISP, PatchDock and FireDock, we predicted that the two domains may interact with each other through electrostatic interactions between at least six pairs of amino acids. This N and C-domains interacted with each other in 1:1 ratio and the hybrid lysed mouse erythrocytes in a slower kinetics when compared with wild type native Cp-PLC. BALB/c mice when challenged with N and C-domain hybrid demonstrated severe myonecrosis at the site of injection while no death was observed. Our results provide further insight into better understanding the mechanism for the toxicity of Cp-PLC N and C-domain mixture.

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Dot Far-Western Blot Analysis of Relative Binding Affinities of the Src Homology 3 Domains of Efs and Its Related Proteins

Cited by 14 publications

References 28 publications

AglZ Is a Filament-Forming Coiled-Coil Protein Required for Adventurous Gliding Motility ofMyxococcus xanthus

AglZ Is a Filament-Forming Coiled-Coil Protein Required for Adventurous Gliding Motility ofMyxococcus xanthus

FCCC Institutional Breast Cancer Training Program

In Silico, In Vitro and In Vivo Analysis of Binding Affinity between N and C-Domains of Clostridium perfringens Alpha Toxin

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