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Find video protocols related to scientific articles indexed in Pubmed.
A PH Domain in ACAP1 Possesses Key Features of the BAR Domain in Promoting Membrane Curvature.
Dev. Cell
PUBLISHED: 06-10-2014
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The BAR (Bin-Amphiphysin-Rvs) domain undergoes dimerization to produce a curved protein structure, which superimposes onto membrane through electrostatic interactions to sense and impart membrane curvature. In some cases, a BAR domain also possesses an amphipathic helix that inserts into the membrane to induce curvature. ACAP1 (Arfgap with Coil coil, Ankyrin repeat, and PH domain protein 1) contains a BAR domain. Here, we show that this BAR domain can neither bind membrane nor impart curvature, but instead requires a neighboring PH (Pleckstrin Homology) domain to achieve these functions. Specific residues within the PH domain are responsible for both membrane binding and curvature generation. The BAR domain adjacent to the PH domain instead interacts with the BAR domains of neighboring ACAP1 proteins to enable clustering at the membrane. Thus, we have uncovered the molecular basis for an unexpected and unconventional collaboration between PH and BAR domains in membrane bending.
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Molecular insights into the membrane-associated phosphatidylinositol 4-kinase II?.
Nat Commun
PUBLISHED: 03-05-2014
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Phosphatidylinositol 4-kinase II? (PI4KII?), a membrane-associated PI kinase, plays a central role in cell signalling and trafficking. Its kinase activity critically depends on palmitoylation of its cysteine-rich motif (-CCPCC-) and is modulated by the membrane environment. Lack of atomic structure impairs our understanding of the mechanism regulating kinase activity. Here we present the crystal structure of human PI4KII? in ADP-bound form. The structure identifies the nucleotide-binding pocket that differs notably from that found in PI3Ks. Two structural insertions, a palmitoylation insertion and an RK-rich insertion, endow PI4KII? with the 'integral' membrane-binding feature. Molecular dynamics simulations, biochemical and mutagenesis studies reveal that the palmitoylation insertion, containing an amphipathic helix, contributes to the PI-binding pocket and anchors PI4KII? to the membrane, suggesting that fluctuation of the palmitoylation insertion affects PI4KII?'s activity. We conclude from our results that PI4KII?'s activity is regulated indirectly through changes in the membrane environment.
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Revealing various coupling of electron transfer and proton pumping in mitochondrial respiratory chain.
Curr. Opin. Struct. Biol.
PUBLISHED: 05-07-2013
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Cellular respiration is the process that releases energy from food and supplies energy for life processes. The mitochondrial respiratory chain is the final and most important step for cellular respiration and is located on the inner membrane of mitochondrion and comprises four large trans-membrane protein complexes (respiratory chain Complexes I, II, III and IV) as well as ubiquinone between Complexes I/II and III and cytochrome c between Complexes III and IV. The function of mitochondrial respiratory chain is biological oxidation by transferring electrons from NADH and succinate to oxygen and then generating proton gradient across the inner membrane. Such proton gradient is utilized by ATP synthase (ATPase, also called as Complex V) to produce energy molecules ATP. Structural studies of mitochondrial respiratory membrane protein complexes are important to understand the mechanism of electron transfer and the redox-coupled proton translocation across the inner membrane. Here, according to the time line, we reviewed the great achievements on structural studies of mitochondrial respiratory complexes in the past twenty years as well as the recent research progresses on the structures of mitochondrial respiratory supra-complexes.
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Flexible interwoven termini determine the thermal stability of thermosomes.
Protein Cell
PUBLISHED: 04-06-2013
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Group II chaperonins, which assemble as double-ring complexes, assist in the refolding of nascent peptides or denatured proteins in an ATP-dependent manner. The molecular mechanism of group II chaperonin assembly and thermal stability is yet to be elucidated. Here, we selected the group II chaperonins (cpn-? and cpn-?), also called thermosomes, from Acidianus tengchongensis and investigated their assembly and thermal stability. We found that the binding of ATP or its analogs contributed to the successful assembly of thermosomes and enhanced their thermal stabilities. Cpn-? is more thermally stable than cpn-?, while the thermal stability of the hetero thermosome cpn-?? is intermediate. Cryo-electron microscopy reconstructions of cpn-? and cpn-? revealed the interwoven densities of their non-conserved flexible N/C-termini around the equatorial planes. The deletion or swapping of their termini and pH-dependent thermal stability assays revealed the key role of the termini electrostatic interactions in the assembly and thermal stability of the thermosomes.
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Thiabendazole inhibits ubiquinone reduction activity of mitochondrial respiratory complex II via a water molecule mediated binding feature.
Protein Cell
PUBLISHED: 06-29-2011
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The mitochondrial respiratory complex II or succinate: ubiquinone oxidoreductase (SQR) is a key membrane complex in both the tricarboxylic acid cycle and aerobic respiration. Five disinfectant compounds were investigated with their potent inhibition effects on the ubiquinone reduction activity of the porcine mitochondrial SQR by enzymatic assay and crystallography. Crystal structure of the SQR bound with thiabendazole (TBZ) reveals a different inhibitor-binding feature at the ubiquinone binding site where a water molecule plays an important role. The obvious inhibitory effect of TBZ based on the biochemical data (IC(50) ~100 ?mol/L) and the significant structure-based binding affinity calculation (~94 ?mol/L) draw the suspicion of using TBZ as a good disinfectant compound for nematode infections treatment and fruit storage.
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Autotransporter passenger domain secretion requires a hydrophobic cavity at the extracellular entrance of the ?-domain pore.
Biochem. J.
PUBLISHED: 02-11-2011
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Whooping cough (pertussis) is a highly contagious acute respiratory illness of humans caused by the Gram-negative bacterial pathogen Bordetella pertussis. The AT (autotransporter) BrkA (Bordetella serum-resistance killing protein A) is an important B. pertussis virulence factor that confers serum resistance and mediates adherence. In the present study, we have solved the crystal structure of the BrkA ?-domain at 3 Å (1 Å=0.1 nm) resolution. Special features are a hairpin-like structure formed by the external loop L4, which is observed fortuitously sitting inside the pore of the crystallographic adjacent ?-domain, and a previously undiscovered hydrophobic cavity formed by patches on loop L4 and ?-strands S5 and S6. This adopts a ubiquitous structure characteristic of all AT ?-domains. Mutagenesis studies have demonstrated that the hairpin-like structure and hydrophobic cavity are crucial for BrkA passenger domain (virulence effector) translocation. This structure helps in understanding the molecular mechanism of AT assembly and secretion and provides a potential target for anti-pertussis drug design.
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Crystal structure of group II chaperonin in the open state.
Structure
PUBLISHED: 06-01-2010
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Thermosomes are group II chaperonins responsible for protein refolding in an ATP-dependent manner. Little is known regarding the conformational changes of thermosomes during their functional cycle due to a lack of high-resolution structure in the open state. Here, we report the first complete crystal structure of thermosome (rATcpn?) in the open state from Acidianus tengchongensis. There is a ?30° rotation of the apical and lid domains compared with the previous closed structure. Besides, the structure reveals a conspicuous hydrophobic patch in the lid domain, and residues locating in this patch are conserved across species. Both the closed and open forms of rATcpn? were also reconstructed by electron microscopy (EM). Structural fitting revealed the detailed conformational change from the open to the closed state. Structural comparison as well as protease K digestion indicated only ATP binding without hydrolysis does not induce chamber closure of thermosome.
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Expression, purification and preliminary biochemical studies of the N-terminal domain of leucine-rich repeat kinase 2.
Biochim. Biophys. Acta
PUBLISHED: 05-04-2010
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Leucine-rich repeat kinase 2 gene is a key factor for Parkinsons disease and encodes for a large protein kinase LRRK2 (280kDa) with multiple domains, including the different repeat sequences at the N-terminus such as ankyrin domain. Here, we successfully expressed and purified two kinds of LRRK2s N-terminal fragments N1 (aa12-320) and N2 (aa12-860). The purified N2 protein was identified by mass spectrometry and N1s molecular weight was determined to be 33.23kDa. Gel filtration revealed that N1 exhibits as monomer, dimer and tetramer and N2 as oligomer in solution. N1s multiple oligomeric states were further proved by native-page and cross-linking gel experiments. Circular dichroism spectrum indicated that N1 and N2 contain both alpha helixes and beta sheets. The polymerization character of LRRK2 N-terminal region would be speculated to relate with its biological function.
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Mechanistic insights into regulated cargo binding by ACAP1 protein.
J. Biol. Chem.
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Coat complexes sort protein cargoes into vesicular transport pathways. An emerging class of coat components has been the GTPase-activating proteins (GAPs) that act on the ADP-ribosylation factor (ARF) family of small GTPases. ACAP1 (ArfGAP with coiled-coil, ankyrin repeat, and PH domains protein 1) is an ARF6 GAP that also acts as a key component of a recently defined clathrin complex for endocytic recycling. Phosphorylation by Akt has been shown to enhance cargo binding by ACAP1 in explaining how integrin recycling is an example of regulated transport. We now shed further mechanistic insights into how this regulation is achieved at the level of cargo binding by ACAP1. We initially defined a critical sequence in the cytoplasmic domain of integrin ?1 recognized by ACAP1 and showed that this sequence acts as a recycling sorting signal. We then pursued a combination of structural, modeling, and functional studies, which suggest that phosphorylation of ACAP1 relieves a localized mechanism of autoinhibition in regulating cargo binding. Thus, we have elucidated a key regulatory juncture that controls integrin recycling and also advanced the understanding of how regulated cargo binding can lead to regulated transport.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.