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Find video protocols related to scientific articles indexed in Pubmed.
Protein Hit1, a novel box C/D snoRNP assembly factor, controls cellular concentration of the scaffolding protein Rsa1 by direct interaction.
Nucleic Acids Res.
PUBLISHED: 08-28-2014
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Biogenesis of eukaryotic box C/D small nucleolar ribonucleoprotein particles (C/D snoRNPs) involves conserved trans-acting factors, which are proposed to facilitate the assembly of the core proteins Snu13p/15.5K, Nop58p/NOP58, Nop56p/NOP56 and Nop1p/Fibrillarin on box C/D small nucleolar RNAs (C/D snoRNAs). In yeast, protein Rsa1 acts as a platform, interacting with both the RNA-binding core protein Snu13 and protein Pih1 of the Hsp82-R2TP chaperone complex. In this work, a proteomic approach coupled with functional and structural studies identifies protein Hit1 as a novel Rsa1p-interacting partner involved in C/D snoRNP assembly. Hit1p contributes to in vivo C/D snoRNA stability and pre-RNA maturation kinetics. It associates with U3 snoRNA precursors and influences its 3'-end processing. Remarkably, Hit1p is required to maintain steady-state levels of Rsa1p. This stabilizing activity is likely to be general across eukaryotic species, as the human protein ZNHIT3(TRIP3) showing sequence homology with Hit1p regulates the abundance of NUFIP1, the Rsa1p functional homolog. The nuclear magnetic resonance solution structure of the Rsa1p317-352-Hit1p70-164 complex reveals a novel mode of protein-protein association explaining the strong stability of the Rsa1p-Hit1p complex. Our biochemical data show that C/D snoRNAs and the core protein Nop58 can interact with the purified Snu13p-Rsa1p-Hit1p heterotrimer.
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In vivo tmRNA protection by SmpB and pre-ribosome binding conformation in solution.
RNA
PUBLISHED: 08-18-2014
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TmRNA is an abundant RNA in bacteria with tRNA and mRNA features. It is specialized in trans-translation, a translation rescuing system. We demonstrate that its partner protein SmpB binds the tRNA-like region (TLD) in vivo and chaperones the fold of the TLD-H2 region. We use an original approach combining the observation of tmRNA degradation pathways in a heterologous system, the analysis of the tmRNA digests by MS and NMR, and co-overproduction assays of tmRNA and SmpB. We study the conformation in solution of tmRNA alone or in complex with one SmpB before ribosome binding using SAXS. Our data show that Mg(2+) drives compaction of the RNA structure and that, in the absence of Mg(2+), SmpB has a similar effect albeit to a lesser extent. Our results show that tmRNA is intrinsically structured in solution with identical topology to that observed on complexes on ribosomes which should facilitate its subsequent recruitment by the 70S ribosome, free or preloaded with one SmpB molecule.
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Functional and structural characterization of 2-amino-4-phenylthiazole inhibitors of the HIV-1 nucleocapsid protein with antiviral activity.
ACS Chem. Biol.
PUBLISHED: 07-07-2014
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The nucleocapsid protein (NC) is a highly conserved protein in diverse HIV-1 subtypes that plays a central role in virus replication, mainly by interacting with conserved nucleic acid sequences. NC is considered a highly profitable drug target to inhibit multiple steps in the HIV-1 life cycle with just one compound, a unique property not shown by any of the other antiretroviral classes. However, most of NC inhibitors developed so far act through an unspecific and potentially toxic mechanism (zinc ejection) and are mainly being investigated as topical microbicides. In an effort to provide specific NC inhibitors that compete for the binding of nucleic acids to NC, here we combined molecular modeling, organic synthesis, biophysical studies, NMR spectroscopy, and antiviral assays to design, synthesize, and characterize an efficient NC inhibitor endowed with antiviral activity in vitro, a desirable property for the development of efficient antiretroviral lead compounds.
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Supramolecular luminescent lanthanide dimers for fluoride sequestering and sensing.
Angew. Chem. Int. Ed. Engl.
PUBLISHED: 04-30-2014
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Lanthanide complexes (Ln=Eu, Tb, and Yb) that are based on a C2 -symmetric cyclen scaffold were prepared and characterized. The addition of fluoride anions to aqueous solutions of the complexes resulted in the formation of dinuclear supramolecular compounds in which the anion is confined into the cavity that is formed by the two complexes. The supramolecular assembly process was monitored by UV/Vis absorption, luminescence, and NMR spectroscopy and high-resolution mass spectrometry. The X-ray crystal structure of the europium dimer revealed that the architecture of the scaffold is stabilized by synergistic effects of the Eu?F?Eu bridging motive, ??stacking interactions, and a four-component hydrogen-bonding network, which control the assembly of the two [EuL] entities around the fluoride ion. The strong association in water allowed for the luminescence sensing of fluoride down to a detection limit of 24?nM.
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Hot-spot analysis to dissect the functional protein-protein interface of a tRNA-modifying enzyme.
Proteins
PUBLISHED: 01-31-2014
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Interference with protein-protein interactions of interfaces larger than 1500 Å(2) by small drug-like molecules is notoriously difficult, particularly if targeting homodimers. The tRNA modifying enzyme Tgt is only functionally active as a homodimer. Thus, blocking Tgt dimerization is a promising strategy for drug therapy as this protein is key to the development of Shigellosis. Our goal was to identify hot-spot residues which, upon mutation, result in a predominantly monomeric state of Tgt. The detailed understanding of the spatial location and stability contribution of the individual interaction hot-spot residues and the plasticity of motifs involved in the interface formation is a crucial prerequisite for the rational identification of drug-like inhibitors addressing the respective dimerization interface. Using computational analyses, we identified hot-spot residues that contribute particularly to dimer stability: a cluster of hydrophobic and aromatic residues as well as several salt bridges. This in silico prediction led to the identification of a promising double mutant, which was validated experimentally. Native nano-ESI mass spectrometry showed that the dimerization of the suggested mutant is largely prevented resulting in a predominantly monomeric state. Crystal structure analysis and enzyme kinetics of the mutant variant further support the evidence for enhanced monomerization and provide first insights into the structural consequences of the dimer destabilization.
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Launching Spiking Ligands into a Protein-Protein Interface: A Promising Strategy To Destabilize and Break Interface Formation in a tRNA Modifying Enzyme.
ACS Chem. Biol.
PUBLISHED: 05-14-2013
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Apart from competitive active-site inhibition of protein function, perturbance of protein-protein interactions by small molecules in oligodomain enzymes opens new perspectives for innovative therapeutics. tRNA-guanine transglycosylase (TGT), a potential target to treat shigellosis, is active only as the homodimer. Consequently, disruption of the dimer interface by small molecules provides a novel inhibition mode. A special feature of this enzyme is the short distance between active site and rim of the dimer interface. This suggests design of expanded active-site inhibitors decorated with rigid, needle-type substituents to spike into potential hot spots of the interaction interface. Ligands with attached ethinyl-type substituents have been synthesized and characterized by Kd measurements, crystallography, noncovalent mass spectrometry, and computer simulations. In contrast to previously determined crystal structures with nonextended active-site inhibitors, a well-defined loop-helix motif, involved in several contacts across the dimer interface, falls apart and suggests enhanced flexibility once the spiking ligands are bound. Mass spectrometry indicates significant destabilization but not full disruption of the complexed TGT homodimer in solution. As directed interactions of the loop-helix motif obviously do not determine dimer stability, a structurally conserved hydrophobic patch composed of several aromatic amino acids is suggested as interaction hot spot. The residues of this patch reside on a structurally highly conserved helix-turn-helix motif, which remains unaffected by the bound spiking ligands. Nevertheless, it is shielded from solvent access by the loop-helix motif that becomes perturbed upon binding of the spiking ligands, which serves as a possible explanation for reduced interface stability.
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Simultaneous self-assembly of a [2]catenane, a trefoil knot, and a Solomon link from a simple pair of ligands.
Angew. Chem. Int. Ed. Engl.
PUBLISHED: 03-22-2013
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A topological triptych: Three molecular links, a [2]catenane, a trefoil knot, and a Solomon link, were obtained in one pot through the self-assembly of two simple ligands in the presence of Zn(II). The approach relied on dynamic covalent chemistry and metal templation.
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The RYMV-encoded viral suppressor of RNA silencing P1 is a zinc-binding protein with redox-dependent flexibility.
J. Mol. Biol.
PUBLISHED: 03-14-2013
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Viral suppressors of RNA interference (VSRs) target host gene silencing pathways, thereby operating important roles in the viral cycle and in host cells, in which they counteract host innate immune responses. However, the molecular mechanisms of VSRs are poorly understood. We provide here biochemical and biophysical features of the dual suppressor/activator VSR P1 protein encoded by the rice yellow mottle virus. In silico analyses of P1 suggested common features with zinc finger proteins and native mass spectrometry unambiguously confirmed that recombinant P1 binds reversibly two zinc atoms, each with a different strength. Additionally, we demonstrate that the reaction of P1 with H2O2 leads to zinc release, disulfide bond formation, and protein oligomerization. A reversible protein modification by redox alterations has only been described for a limited number of zinc finger proteins and has never been reported for VSRs. Those reported here for P1 might be a general feature of Cys-rich VSRs and could be a key regulatory mechanism for the control of RNA silencing.
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Noncovalent mass spectrometry for the characterization of antibody/antigen complexes.
Methods Mol. Biol.
PUBLISHED: 03-12-2013
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Monoclonal antibodies (mAbs) have taken on an increasing importance for the treatment of various diseases including cancers, immunological disorders, and other pathologies. These large biomolecules display specific structural features, which affect their efficiency and need therefore to be extensively characterized using sensitive and orthogonal analytical techniques. Among them, mass spectrometry (MS) has become the method of choice to study mAb amino acid sequences as well as their posttranslational modifications with the aim of reducing their chemistry, manufacturing, and control liabilities. This chapter will provide the reader with a description of the general approach allowing antibody/antigen systems to be characterized by noncovalent MS. In the present chapter, we describe how recent noncovalent MS technologies are used to characterize immune complexes involving both murine and humanized mAb 6F4 directed against human JAM-A, a newly identified antigenic protein (Ag) over-expressed in tumor cells. We will detail experimental conditions (sample preparation, optimization of instrumental parameters, etc.) required for the detection of noncovalent antibody/antigen complexes by MS. We will then focus on the type and the reliability of the information that we get from noncovalent MS data, with emphasis on the determination of the stoichiometry of antibody/antigen systems. Noncovalent MS appears as an additional supporting technique for therapeutic mAbs lead characterization and development.
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p-Sulfonato-calix[n]arenes inhibit staphylococcal bicomponent leukotoxins by supramolecular interactions.
Biochem. J.
PUBLISHED: 01-04-2013
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PVL (Panton-Valentine leukocidin) and other Staphylococcus aureus ?-stranded pore-forming toxins are important virulence factors involved in various pathologies that are often necrotizing. The present study characterized leukotoxin inhibition by selected SCns (p-sulfonato-calix[n]arenes): SC4, SC6 and SC8. These chemicals have no toxic effects on human erythrocytes or neutrophils, and some are able to inhibit both the activity of and the cell lysis by leukotoxins in a dose-dependent manner. Depending on the type of leukotoxins and SCns, flow cytometry revealed IC50 values of 6-22 ?M for Ca2+ activation and of 2-50 ?M for cell lysis. SCns were observed to affect membrane binding of class S proteins responsible for cell specificity. Electrospray MS and surface plasmon resonance established supramolecular interactions (1:1 stoichiometry) between SCns and class S proteins in solution, but not class F proteins. The membrane-binding affinity of S proteins was Kd=0.07-6.2 nM. The binding ability was completely abolished by SCns at different concentrations according to the number of benzenes (30-300 ?M; SC8>SC6?SC4). The inhibitory properties of SCns were also observed in vivo in a rabbit model of PVL-induced endophthalmitis. These calixarenes may represent new therapeutic avenues aimed at minimizing inflammatory reactions and necrosis due to certain virulence factors.
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Transcription cofactors TRIM24, TRIM28, and TRIM33 associate to form regulatory complexes that suppress murine hepatocellular carcinoma.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 04-29-2011
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TRIM24 (TIF1?), TRIM28 (TIF1?), and TRIM33 (TIF1?) are three related cofactors belonging to the tripartite motif superfamily that interact with distinct transcription factors. TRIM24 interacts with the liganded retinoic acid (RA) receptor to repress its transcriptional activity. Germ line inactivation of TRIM24 in mice deregulates RA-signaling in hepatocytes leading to the development of hepatocellular carcinoma (HCC). Here we show that TRIM24 can be purified as at least two macromolecular complexes comprising either TRIM33 or TRIM33 and TRIM28. Somatic hepatocyte-specific inactivation of TRIM24, TRIM28, or TRIM33 all promote HCC in a cell-autonomous manner in mice. Moreover, HCC formation upon TRIM24 inactivation is strongly potentiated by further loss of TRIM33. These results demonstrate that the TIF1-related subfamily of TRIM proteins interact both physically and functionally to modulate HCC formation in mice.
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Identification of protein partners of the human immunodeficiency virus 1 tat/rev exon 3 leads to the discovery of a new HIV-1 splicing regulator, protein hnRNP K.
RNA Biol
PUBLISHED: 03-01-2011
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HIV-1 pre-mRNA splicing depends upon 4 donor and 8 acceptor sites, which are used in combination to produce more than 40 different mRNAs. The acceptor site A7 plays an essential role for tat and rev mRNA production. The SLS2-A7 stem-loop structure containing site A7 was also proposed to modulate HIV-1 RNA export by the Rev protein. To further characterize nuclear factors involved in these processes, we purified RNP complexes formed by incubation of SLS2-A7 RNA transcripts in HeLa cell nuclear extracts by affinity chromatography and identified 33 associated proteins by nanoLC-MS/MS. By UV cross-linking, immunoselection and EMSA, we showed that, in addition to the well-known hnRNP A1 inhibitor of site A7, nucleolin, hnRNP H and hnRNP K interact directly with SLS2-A7 RNA. Nucleolin binds to a cluster of successive canonical NRE motifs in SLS2-A7 RNA, which is unique in HIV-1 RNA. Proteins hnRNP A1 and hnRNP K bind synergistically to SLS2-A7 RNA and both have a negative effect on site A7 activity. By the use of a plasmid expressing a truncated version of HIV-1 RNA, we showed a strong effect of the overexpression of hnRNP K in HeLa cells on HIV-1 alternative splicing. As a consequence, production of the Nef protein was strongly reduced. Interestingly also, many proteins identified in our proteomic analysis are known to modulate either the Rev activity or other mechanisms required for HIV-1 multiplication and several of them seem to be recruited by hnRNP K, suggesting that hnRNP K plays an important role for HIV-1 biology.
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Poly(ADP-ribose) polymerase 3 (PARP3), a newcomer in cellular response to DNA damage and mitotic progression.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 01-26-2011
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The ADP ribosyl transferase [poly(ADP-ribose) polymerase] ARTD3(PARP3) is a newly characterized member of the ARTD(PARP) family that catalyzes the reaction of ADP ribosylation, a key posttranslational modification of proteins involved in different signaling pathways from DNA damage to energy metabolism and organismal memory. This enzyme shares high structural similarities with the DNA repair enzymes PARP1 and PARP2 and accordingly has been found to catalyse poly(ADP ribose) synthesis. However, relatively little is known about its in vivo cellular properties. By combining biochemical studies with the generation and characterization of loss-of-function human and mouse models, we describe PARP3 as a newcomer in genome integrity and mitotic progression. We report a particular role of PARP3 in cellular response to double-strand breaks, most likely in concert with PARP1. We identify PARP3 as a critical player in the stabilization of the mitotic spindle and in telomere integrity notably by associating and regulating the mitotic components NuMA and tankyrase 1. Both functions open stimulating prospects for specifically targeting PARP3 in cancer therapy.
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Monitoring the dynamics of monomer exchange using electrospray mass spectrometry: the case of the dimeric glucosamine-6-phosphate synthase.
J. Am. Soc. Mass Spectrom.
PUBLISHED: 01-15-2011
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Escherichia coli glucosamine-6-phosphate synthase (GlmS) is a dimeric enzyme from the glutamine-dependent amidotransferases family, which catalyses the conversion of D-fructose-6-phosphate (Fru6P) and glutamine (Gln) into D-glucosamine-6-phosphate (GlcN6P) and glutamate, respectively. Extensive X-ray crystallography investigations have been reported, highlighting the importance of the dimeric association to form the sugar active site as well as significant conformational changes of the protein upon substrate and product binding. In the present work, an approach based on time-resolved noncovalent mass spectrometry has been developed to study the dynamics of GlmS subunit exchange. Using (14)N versus (15)N labeled proteins, the kinetics of GlmS subunit exchange was monitored with the wild-type enzyme in the presence of different substrates and products as well as with the protein bearing a key amino acid mutation specially designed to weaken the dimer interface. Determination of rate constants of subunit exchange revealed important modifications of the protein dynamics: while glutamine, glutamate, and K603A mutation accelerates subunit exchange, Fru6P and GlcN6P totally prevent it. These results are described in light of the available structural information, providing additional useful data for both the characterization of GlmS catalytic process and the design of new GlmS inhibitors. Finally, time-resolved noncovalent MS can be proposed as an additional biophysical technique for real-time monitoring of protein dynamics.
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Homodimerization of the death-associated protein kinase catalytic domain: development of a new small molecule fluorescent reporter.
PLoS ONE
PUBLISHED: 11-01-2010
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Death-Associated Protein Kinase (DAPK) is a member of the Ca2+/calmodulin regulated serine/threonine protein kinases. Its biological function has been associated with induced cell death, and in vivo use of selective small molecule inhibitors of DAPK catalytic activity has demonstrated that it is a potential therapeutic target for treatment of brain injuries and neurodegenerative diseases.
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The Pih1-Tah1 cochaperone complex inhibits Hsp90 molecular chaperone ATPase activity.
J. Biol. Chem.
PUBLISHED: 07-27-2010
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Hsp90 (heat shock protein 90) is an ATP-dependent molecular chaperone regulated by collaborating proteins called cochaperones. This machinery is involved in the conformational activation of client proteins like signaling kinases, transcription factors, or ribonucleoproteins (RNP) such as telomerase. TPR (TetratricoPeptide Repeat)-containing protein associated with Hsp90 (Tah1) and protein interacting with Hsp90 (Pih1) have been identified in Saccharomyces cerevisiae as two Hsp90 cochaperones involved in chromatin remodeling complexes and small nucleolar RNP maturation. Tah1 possesses a minimal TPR domain and binds specifically to the Hsp90 C terminus, whereas Pih1 displays no homology to other protein motifs and has been involved in core RNP protein interaction. While Pih1 alone was unstable and was degraded from its N terminus, we showed that Pih1 and Tah1 form a stable heterodimeric complex that regulates Hsp90 ATPase activity. We used different biophysical approaches such as analytical ultracentrifugation, microcalorimetry, and noncovalent mass spectrometry to characterize the Pih1-Tah1 complex and its interaction with Hsp90. We showed that the Pih1-Tah1 heterodimer binds to Hsp90 with a similar affinity and the same stoichiometry as Tah1 alone. However, the Pih1-Tah1 complex antagonizes Tah1 activity on Hsp90 and inhibits the chaperone ATPase activity. We further identified the region within Pih1 responsible for interaction with Tah1 and inhibition of Hsp90, allowing us to suggest an interaction model for the Pih1-Tah1/Hsp90 complex. These results, together with previous reports, suggest a role for the Pih1-Tah1 cochaperone complex in the recruitment of client proteins such as core RNP proteins to Hsp90.
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Physical basis of the inducer-dependent cooperativity of the Central glycolytic genes Repressor/DNA complex.
Nucleic Acids Res.
PUBLISHED: 05-12-2010
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The Central glycolytic genes Repressor (CggR) from Bacillus subtilis belongs to the SorC family of transcription factors that control major carbohydrate metabolic pathways. Recent studies have shown that CggR binds as a tetramer to its tandem operator DNA sequences and that the inducer metabolite, fructose 1,6-bisphosphate (FBP), reduces the binding cooperativity of the CggR/DNA complex. Here, we have determined the effect of FBP on the size, shape and stoichiometry of CggR complexes with full-length and half-site operator sequence by small-angle X-ray scattering, size-exclusion chromatography, ?uorescence cross-correlation spectroscopy and noncovalent mass spectrometry (MS). Our results show that CggR forms a compact tetrameric assembly upon binding to either the full-length operator or two half-site DNAs and that FBP triggers a tetramer-dimer transition that leaves a single dimer on the half-site or two physically independent dimers on the full-length target. Although the binding of other phospho-sugars was evidenced by MS, only FBP was found to completely disrupt dimer-dimer contacts. We conclude that inducer-dependent dimer-dimer bridging interactions constitute the physical basis for CggR cooperative binding to DNA and the underlying repression mechanism. This work provides experimental evidences for a cooperativity-based regulation model that should apply to other SorC family members.
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Analysis of recombinant phosphoprotein complexes with complementary mass spectrometry approaches.
Anal. Biochem.
PUBLISHED: 04-15-2010
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The baculovirus expression vector system is recognized as a powerful and versatile tool for producing large quantities of recombinant proteins that cannot be obtained in Escherichia coli. Here we report (i) the purification of the recombinant cyclin-dependent kinase (CDK)-activating kinase (CAK) complex, which includes CDK7, cyclin H, and MAT1 proteins, and (ii) the functional characterization of CAK together with a detailed analysis and mapping of the phosphorylation states and sites using mass spectrometry (MS). In vitro kinase assay showed that recombinant CAK is able to phosphorylate the cyclin-dependent kinase CDK2 implicated in cell cycle progression and the carboxy-terminal domain (CTD) of the eukaryotic RNA polymerase II. An original combination of MS techniques was used for the determination of the phosphorylation sites of each constitutive subunit at both protein and peptide levels. Liquid chromatography (LC)-MS analysis of intact proteins demonstrated that none of the CAK subunits was fully modified and that the phosphorylation pattern of recombinant CAK is extremely heterogeneous. Finally, matrix-assisted laser desorption/ionization (MALDI)-MS and nanoLC-tandem mass spectrometry (MS/MS) techniques were used for the analysis of the major phosphorylation sites of each subunit, showing that all correspond to Ser/Thr phosphorylation sites. Phosphorylations occurred on Ser164 and Thr170 residues of CDK7, Thr315 residue of cyclin H, and Ser279 residue of MAT1.
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Combination of noncovalent mass spectrometry and traveling wave ion mobility spectrometry reveals sugar-induced conformational changes of central glycolytic genes repressor/DNA complex.
Anal. Chem.
PUBLISHED: 04-06-2010
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The central glycolytic genes repressor (CggR) is a 37 kDa transcriptional repressor protein which plays a key role in Bacillus subtilis glycolysis by regulating the transcription of the gapA operon. Fructose-1,6-bisphosphate (FBP), identified as the effector sugar, has been shown to abolish the binding cooperativity of CggR to its DNA target and to modify the conformational dynamics of the CggR/DNA complex. In the present study, noncovalent mass spectrometry (MS) was used to obtain deeper insights into FBP-dependent CggR/DNA interactions. The effect of FBP binding on CggR alone and on CggR/DNA complexes was examined using automated chip-based nanoelectrospray MS and traveling wave ion mobility mass spectrometry (IM-MS). Our results revealed that tetrameric CggR dissociates into dimers upon FBP binding. Moreover, FBP binding to CggR/DNA complexes triggers disruption of intermolecular protein/protein interactions within the complex, significantly modifying its conformation as evidenced by a 5% increase of its collision cross section. For the first time, the use of IM-MS is reported to probe ligand-induced conformational modifications of a protein/DNA complex with an emphasis on the comparison with solution-based techniques.
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2-D structure of the A region of Xist RNA and its implication for PRC2 association.
PLoS Biol.
PUBLISHED: 01-05-2010
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In placental mammals, inactivation of one of the X chromosomes in female cells ensures sex chromosome dosage compensation. The 17 kb non-coding Xist RNA is crucial to this process and accumulates on the future inactive X chromosome. The most conserved Xist RNA region, the A region, contains eight or nine repeats separated by U-rich spacers. It is implicated in the recruitment of late inactivated X genes to the silencing compartment and likely in the recruitment of complex PRC2. Little is known about the structure of the A region and more generally about Xist RNA structure. Knowledge of its structure is restricted to an NMR study of a single A repeat element. Our study is the first experimental analysis of the structure of the entire A region in solution. By the use of chemical and enzymatic probes and FRET experiments, using oligonucleotides carrying fluorescent dyes, we resolved problems linked to sequence redundancies and established a 2-D structure for the A region that contains two long stem-loop structures each including four repeats. Interactions formed between repeats and between repeats and spacers stabilize these structures. Conservation of the spacer terminal sequences allows formation of such structures in all sequenced Xist RNAs. By combination of RNP affinity chromatography, immunoprecipitation assays, mass spectrometry, and Western blot analysis, we demonstrate that the A region can associate with components of the PRC2 complex in mouse ES cell nuclear extracts. Whilst a single four-repeat motif is able to associate with components of this complex, recruitment of Suz12 is clearly more efficient when the entire A region is present. Our data with their emphasis on the importance of inter-repeat pairing change fundamentally our conception of the 2-D structure of the A region of Xist RNA and support its possible implication in recruitment of the PRC2 complex.
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Crystal growth of proteins, nucleic acids, and viruses in gels.
Prog. Biophys. Mol. Biol.
PUBLISHED: 11-27-2009
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Medium-sized single crystals with perfect habits and no defect producing intense and well-resolved diffraction patterns are the dream of every protein crystallographer. Crystals of biological macromolecules possessing these characteristics can be prepared within a medium in which mass transport is restricted to diffusion. Chemical gels (like polysiloxane) and physical gels (such as agarose) provide such an environment and are therefore suitable for the crystallisation of biological macromolecules. Instructions for the preparation of each type of gel are given to urge crystal growers to apply diffusive media for enhancing crystallographic quality of their crystals. Examples of quality enhancement achieved with silica and agarose gels are given. Results obtained with other substances forming gel-like media (such as lipidic phases and cellulose derivatives) are presented. Finally, the use of gels in combination with capillary tubes for counter-diffusion experiments is discussed. Methods and techniques implemented with proteins can also be applied to nucleic acids and nucleoprotein assemblies such as viruses.
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Extending mass spectrometry contribution to therapeutic monoclonal antibody lead optimization: characterization of immune complexes using noncovalent ESI-MS.
Anal. Chem.
PUBLISHED: 07-11-2009
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Monoclonal antibodies (mAbs) have taken on an increasing importance for the treatment of various diseases including cancers, immunological disorders, and other pathologies. These large biomolecules display specific structural features, which affect their efficiency and need, therefore, to be extensively characterized using sensitive and orthogonal analytical techniques. Among them, mass spectrometry (MS) has become the method of choice to study mAb amino acid sequences as well as their post-translational modifications. In the present work, recent noncovalent MS-technologies including automated chip-based nanoelectrospray MS and traveling wave ion mobility MS were used for the first time to characterize immune complexes involving both murine and humanized mAb 6F4 directed against human JAM-A, a newly identified antigenic protein (Ag) overexpressed in tumor cells. MS-based structural insights evidenced that heterogeneous disulfide bridge pairings of recombinant JAM-A alter neither its native structure nor mAbs 6F4 recognition properties. Investigations focused on mAb:Ag complexes revealed that, similarly to murine mAb, humanized mAb 6F4 binds selectively up to four antigen molecules with a similar affinity, confirming in this way the reliability of the humanization process. Noncovalent MS appears as an additional supporting technique for therapeutic mAbs lead characterization and development.
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Characterization of human and bovine phosphatidylethanolamine-binding protein (PEBP/RKIP) interactions with morphine and morphine-glucuronides determined by noncovalent mass spectrometry.
Med. Sci. Monit.
PUBLISHED: 07-01-2009
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The phosphatidylethanolamine-binding protein (PEBP/RKIP), initially found to bind phosphatidylethanolamine (PE), has been shown to be associated with morphine derivatives. Our recent study on bovine primary chromaffin cells showed that inside secretory granules, PEBP is noncovalently associated to endogenous morphine-6-glucuronide (M6G), a highly analgesic morphine metabolite. During stress, M6G-PEBP complexes may be released into circulation to target peripheral opioid receptors. We now report the investigation of PEBP binding properties towards morphine and morphine analogs.
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An integrative approach combining noncovalent mass spectrometry, enzyme kinetics and X-ray crystallography to decipher Tgt protein-protein and protein-RNA interaction.
J. Mol. Biol.
PUBLISHED: 05-25-2009
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The tRNA-modifying enzyme tRNA-guanine transglycosylase (Tgt) is a putative target for new selective antibiotics against Shigella bacteria. The formation of a Tgt homodimer was suggested on the basis of several crystal structures of Tgt in complex with RNA. In the present study, noncovalent mass spectrometry was used (i) to confirm the dimeric oligomerization state of Tgt in solution and (ii) to evidence the binding stoichiometry of the complex formed between Tgt and its full-length substrate tRNA. To further investigate the importance of Tgt protein-protein interaction, point mutations were introduced into the dimer interface in order to study their influence on the formation of the catalytically active complex. Enzyme kinetics revealed a reduced catalytic activity of these mutated variants, which could be related to a destabilization of the dimer formation as evidenced by both noncovalent mass spectrometry and X-ray crystallography. Finally, the effect of inhibitor binding was investigated by noncovalent mass spectrometry, thus providing the binding stoichiometries of Tgt:inhibitor complexes and showing competitive interactions in the presence of tRNA. Inhibitors that display an influence on the formation of the dimer interface in the crystal structure are promising candidates to alter the protein-protein interaction, which could provide a new way to inhibit Tgt.
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Proteomic analysis of malignant B-cell derived microparticles reveals CD148 as a potentially useful antigenic biomarker for mantle cell lymphoma diagnosis.
J. Proteome Res.
PUBLISHED: 05-06-2009
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The diagnosis of mature B-cell neoplasms (MBCN) remains difficult in a number of cases, especially leukemic phases of non-Hodgkin lymphoma, for which discriminating criteria or biomarker are often lacking. To identify new surface biomarkers, we developed an original proteomic approach based on mass spectrometry analysis of plasma membrane microparticles derived from chronic B-cell lymphoproliferations of single patients: chronic lymphocytic leukemia (CLL), small cell lymphoma (SLL) and mantle cell lymphoma (MCL). A straightforward selection process for proteomic-based candidate biomarker identification was further constructed in order to propose potentially useful and relevant biomarkers. Comparison of the lists of the proteins identified in each pathology combined to highly stringent MS validation criteria for protein identification allowed to propose CD148, a membrane receptor with phosphatase activity, as a discriminating biomarker candidate. Flow cytometry analyses, performed on 158 patients and 30 controls, showed that an anti-CD148 antibody stained significantly higher MCL than CLL and SLL circulating cells (p<0.0001), which validates CD148 overexpression in MCL. Our results indicate that a medium or high CD148 expression level may exclude the diagnosis of CLL and high CD148 expression levels (CD148 MFI equal or superior to 2 times the value obtained with CLL/SLL) allows MCL diagnosis to be suspected with 91% specificity (versus CLL and SLL) and 78% sensitivity. This study is one of the first where proteomic strategies allowed to identify a potentially useful biomarker.
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Structural and functional insights into the ligand-binding domain of a nonduplicated retinoid X nuclear receptor from the invertebrate chordate amphioxus.
J. Biol. Chem.
PUBLISHED: 03-13-2009
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Retinoid X nuclear receptors (RXRs), as well as their insect orthologue, ultraspiracle protein (USP), play an important role in the transcription regulation mediated by the nuclear receptors as the common partner of many other nuclear receptors. Phylogenetic and structural studies have shown that the several evolutionary shifts have modified the ligand binding ability of RXRs. To understand the vertebrate-specific character of RXRs, we have studied the RXR ligand-binding domain of the cephalochordate amphioxus (Branchiostoma floridae), an invertebrate chordate that predates the genome duplication that produced the three vertebrates RXRs (alpha, beta, and gamma). Here we report the crystal structure of a novel apotetramer conformation of the AmphiRXR ligand-binding domain, which shows some similarity with the structures of the arthropods RXR/USPs. AmphiRXR adopts an apo antagonist conformation with a peculiar conformation of helix H11 filling the binding pocket. In contrast to the arthropods RXR/USPs, which cannot be activated by any RXR ligands, our functional data show that AmphiRXR, like the vertebrates/mollusk RXRs, is able to bind and be activated by RXR ligands but less efficiently than vertebrate RXRs. Our data suggest that amphioxus RXR is, functionally, an intermediate between arthropods RXR/USPs and vertebrate RXRs.
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Differential action on coregulator interaction defines inverse retinoid agonists and neutral antagonists.
Chem. Biol.
PUBLISHED: 03-11-2009
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Retinoic acid receptors (RARs) are ligand-dependent transcription factors that control a plethora of physiological processes. RARs exert their functions by regulating gene networks controlling cell growth, differentiation, survival, and death. Uncovering the molecular details by which synthetic ligands direct specificity and functionality of nuclear receptors is key to rational drug development. Here we define the molecular basis for (E)-4-[2-[5,6-Dihydro-5,5-dimethyl-8-(2-phenylethynyl)naphthalen-2-yl]ethen-1-yl]benzoic acid (BMS204,493) acting as the inverse pan-RAR agonist and define 4-[5,6-Dihydro-5,5-dimethyl-8-(quinolin-3-yl)naphthalen-2-carboxamido]benzoic acid (BMS195,614) as the neutral RARalpha-selective antagonist. We reveal the details of the differential coregulator interactions imposed on the receptor by the ligands and show that the anchoring of H12 is fundamentally distinct in the presence of the two ligands, thus accounting for the observed effects on coactivator and corepressor interactions. These ligands will facilitate studies on the role of the constitutive activity of RARs, particularly of the tumor suppressor RARbeta, whose specific functions relative to other RARs have remained elusive.
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Detection and structural features of the betaB2-B3-crystallin heterodimer by radical probe mass spectrometry (RP-MS).
J Mass Spectrom
PUBLISHED: 02-12-2009
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The predilection of the beta-crystallin B2 subunit to interact with the betaB3 subunit rather than self associate is evident by the detection of the betaB2-B3-crystallin heterodimer by native gel electrophoresis and electrospray ionisation time-of-flight (ESI-TOF) mass spectrometry under non denaturing conditions. The complex has been detected for the first time and its molecular mass is measured to be 47,450 +/- 1 Da. Radical probe mass spectrometry (RP-MS) was subsequently applied to investigate the nature of the heterodimer through the limited oxidation of the subunits in the complex. Two peptide segments of the betaB2 subunit and six of the betaB3 subunit were found to oxidise, with far greater oxidation observed within the betaB3 versus the betaB2 subunit. This, and the observation that the oxidation data of betaB2 subunit is inconsistent with the structure of the betaB2 monomer, demonstrates that the protection of betaB2 is conferred by its association with betaB3 subunit within the heterodimer where only the residues of, and towards, its N-terminal domain remain exposed to solvent. The results suggest that the betaB2 subunit adopts a more compacted form than in its monomeric form in order for much of its structure to be enveloped by the betaB3 subunit within the heterodimer.
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Identification by high throughput screening of small compounds inhibiting the nucleic acid destabilization activity of the HIV-1 nucleocapsid protein.
Biochimie
PUBLISHED: 01-21-2009
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Due to its highly conserved zinc fingers and its nucleic acid chaperone properties which are critical for HIV-1 replication, the nucleocapsid protein (NC) constitutes a major target in AIDS therapy. Different families of molecules targeting NC zinc fingers and/or inhibiting the binding of NC with its target nucleic acids have been developed. However, their limited specificity and their cellular toxicity prompted us to develop a screening assay to target molecules able to inhibit NC chaperone properties, and more specifically the initial NC-promoted destabilization of the nucleic acid secondary structure. Since this destabilization is critically dependent on the properly folded fingers, the developed assay is thought to be highly specific. The assay was based on the use of cTAR DNA, a stem-loop sequence complementary to the transactivation response element, doubly labelled at its 5 and 3 ends by a rhodamine 6G fluorophore and a fluorescence quencher, respectively. Addition of NC(12-55), a peptide corresponding to the zinc finger domain of NC, to this doubly-labelled cTAR, led to a partial melting of the cTAR stem, which increases the distance between the two labels and thus, restores the rhodamine 6G fluorescence. Thus, positive hits were detected through the decrease of rhodamine 6G fluorescence. An "in-house" chemical library of 4800 molecules was screened and five compounds with IC(50) values in the micromolar range have been selected. The hits were shown by mass spectrometry and fluorescence anisotropy titration to prevent binding of NC(12-55) to cTAR through direct interaction with the NC folded fingers, but without promoting zinc ejection. These non-zinc ejecting NC binders are a new series of anti-NC molecules that could be used to rationally design molecules with potential anti-viral activities.
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A phenyl-thiadiazolylidene-amine derivative ejects zinc from retroviral nucleocapsid zinc fingers and inactivates HIV virions.
Retrovirology
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Sexual acquisition of the human immunodeficiency virus (HIV) through mucosal transmission may be prevented by using topically applied agents that block HIV transmission from one individual to another. Therefore, virucidal agents that inactivate HIV virions may be used as a component in topical microbicides.
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Structural and functional studies of ReP1-NCXSQ, a protein regulating the squid nerve Na+/Ca2+ exchanger.
Acta Crystallogr. D Biol. Crystallogr.
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The protein ReP1-NCXSQ was isolated from the cytosol of squid nerves and has been shown to be required for MgATP stimulation of the squid nerve Na(+)/Ca(2+) exchanger NCXSQ1. In order to determine its mode of action and the corresponding biologically active ligand, sequence analysis, crystal structures and mass-spectrometric studies of this protein and its Tyr128Phe mutant are reported. Sequence analysis suggests that it belongs to the CRABP family in the FABP superfamily. The X-ray structure at 1.28 Å resolution shows the FABP ?-barrel fold, with a fatty acid inside the barrel that makes a relatively short hydrogen bond to Tyr128 and shows a double bond between C9 and C10 but that is disordered beyond C12. Mass-spectrometric studies identified this fatty acid as palmitoleic acid, confirming the double bond between C9 and C10 and establishing a length of 16 C atoms in the aliphatic chain. This acid was caught inside during the culture in Escherichia coli and therefore is not necessarily linked to the biological activity. The Tyr128Phe mutant was unable to activate the Na(+)/Ca(2+) exchanger and the corresponding crystal structure showed that without the hydrogen bond to Tyr128 the palmitoleic acid inside the barrel becomes disordered. Native mass-spectrometric analysis confirmed a lower occupancy of the fatty acid in the Tyr128Phe mutant. The correlation between (i) the lack of activity of the Tyr128Phe mutant, (ii) the lower occupancy/disorder of the bound palmitoleic acid and (iii) the mass-spectrometric studies of ReP1-NCXSQ suggests that the transport of a fatty acid is involved in regulation of the NCXSQ1 exchanger, providing a novel insight into the mechanism of its regulation. In order to identify the biologically active ligand, additional high-resolution mass-spectrometric studies of the ligands bound to ReP1-NCXSQ were performed after incubation with squid nerve vesicles both with and without MgATP. These studies clearly identified palmitic acid as the fatty acid involved in regulation of the Na(+)/Ca(2+) exchanger from squid nerve.
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Crystal packing modifies ligand binding affinity: the case of aldose reductase.
Proteins
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The relationship between the structures of protein-ligand complexes existing in the crystal and in solution, essential in the case of fragment-based screening by X-ray crystallography (FBS-X), has been often an object of controversy. To address this question, simultaneous co-crystallization and soaking of two inhibitors with different ratios, Fidarestat (FID; K(d) = 6.5 nM) and IDD594 (594; K(d) = 61 nM), which bind to h-aldose reductase (AR), have been performed. The subatomic resolution of the crystal structures allows the differentiation of both inhibitors, even when the structures are almost superposed. We have determined the occupation ratio in solution by mass spectrometry (MS) Occ(FID)/Occ(594) = 2.7 and by X-ray crystallography Occ(FID)/Occ(594) = 0.6. The occupancies in the crystal and in solution differ 4.6 times, implying that ligand binding potency is influenced by crystal contacts. A structural analysis shows that the Loop A (residues 122-130), which is exposed to the solvent, is flexible in solution, and is involved in packing contacts within the crystal. Furthermore, inhibitor 594 contacts the base of Loop A, stabilizing it, while inhibitor FID does not. This is shown by the difference in B-factors of the Loop A between the AR-594 and AR-FID complexes. A stable loop diminishes the entropic energy barrier to binding, favoring 594 versus FID. Therefore, the effect of the crystal environment should be taken into consideration in the X-ray diffraction analysis of ligand binding to proteins. This conclusion highlights the need for additional methodologies in the case of FBS-X to validate this powerful screening technique, which is widely used.
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Use of virtual screening for discovering antiretroviral compounds interacting with the HIV-1 nucleocapsid protein.
Virus Res.
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The HIV-1 nucleocapsid protein (NC) is considered as an emerging drug target for the therapy of AIDS. Several studies have highlighted the crucial role of NC within the viral replication cycle. However, although NC inhibition has provided in vitro and in vivo antiretroviral activity, drug-candidates which interfere with NC functions are still missing in the therapeutic arsenal against HIV. Based on previous studies, where the dynamic behavior of NC and its ligand binding properties have been investigated by means of computational methods, here we used a virtual screening protocol for discovering novel antiretroviral compounds which interact with NC. The antiretroviral activity of virtual hits was tested in vitro, whereas biophysical studies elucidated the direct interaction of most active compounds with NC(11-55), a peptide corresponding to the zinc finger domain of NC. Two novel antiretroviral small molecules capable of interacting with NC are presented here.
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Exploring key parameters to detect subtle ligand-induced protein conformational changes using traveling wave ion mobility mass spectrometry.
Anal. Chem.
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Evidencing subtle conformational transitions in proteins occurring upon small modulator binding usually requires atomic resolution techniques (X-ray crystallography or NMR). Recently, hyphenation of ion mobility and mass spectrometry (IM-MS) has greatly enlarged the potentials for biomolecular assembly structural characterization. Using the well 3D-characterized Bcl-xL/ABT-737 protein model, we explored in the present report whether IM-MS can be used to differentiate close conformers and monitor collision cross section (CCS) differences correlating with ligand-induced conformational changes. Because comparing CCS derived from IM-MS data with 3D-computed CCS is critical for thorough data interpretation, discussing pitfalls related to protein construct similarity and missing sequence sections in PDB files was of primary importance to avoid misinterpretation. The methodic exploration of instrument parameters showed enhanced IM separation of Bcl-xL conformers by combining high wave heights and velocities with low helium and nitrogen flow rates while keeping a high He/N(2) flow rate ratio (>3). The robustness of CCS measurements was eventually improved with a modified IM calibration method providing constant CCS values regardless of instrument settings. Altogether, optimized IM-MS settings allowed a 0.4 nm(2) increase (i.e., 2%) of Bcl-xL CCS to be evidenced upon ABT-737 binding.
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Biosimilar, biobetter, and next generation antibody characterization by mass spectrometry.
Anal. Chem.
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This Feature will introduce the strategies of therapeutic antibodies (mAbs) in-depth characterization by mass spectrometry (MS) and discuss analytical comparison of biosimilar to originator mAbs, with the cases of trastuzumab and cetuximab. In addition, the structural and functional insights gained both by state-of-the art and emerging MS methods used for biobetters and next generation antibodies design and optimization will also be highlighted.
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