Sensing peptide–oligonucleotide interactions by a two-color fluorescence label: application to the HIV-1 nucleocapsid protein

Shvadchak, Volodymyr V.; Klymchenko, Andrey S.; Rocquigny, Hugues de; Mély, Yves

doi:10.1093/nar/gkn1083

Cited by 64 publications

(77 citation statements)

References 88 publications

(276 reference statements)

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“…Peptide concentration was determined using an extinction coefficient of 5,700 M Ϫ1 cm Ϫ1 at 280 nm for nonlabeled peptides, 33,000 M Ϫ1 cm Ϫ1 at 400 nm for the MFLlabeled peptides, and 80,000 M Ϫ1 cm Ϫ1 at 555 nm for LRh-labeled NC. Note that the nucleic acid chaperone properties (75,76) of the labeled NC peptide were tested as described previously (72) and found to be close to those of the unlabeled NC, indicating that the MFL label marginally perturbs the peptide structure and activity (data not shown).…”

Section: Methodsmentioning

confidence: 55%

“…NC peptides were prepared by solid-phase peptide synthesis on a 433A synthesizer (ABI, Foster City, CA), HPLC purified, and characterized by ion spray mass spectrometry, as previously described (71,72). The MFL probe, a functionalized derivative of 4=-(dimethylamino)-3-hydroxyflavone, was prepared and coupled to the N terminus of the NC peptides, as previously described (73).…”

Section: Methodsmentioning

confidence: 99%

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The HIV-1 Nucleocapsid Protein Recruits Negatively Charged Lipids To Ensure Its Optimal Binding to Lipid Membranes

Kempf

Postupalenko

Bora

et al. 2015

J Virol

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The HIV-1 Gag polyprotein precursor composed of the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains orchestrates virus assembly via interactions between MA and the cell plasma membrane (PM) on one hand and NC and the genomic RNA on the other hand. As the Gag precursor can adopt a bent conformation, a potential interaction of the NC domain with the PM cannot be excluded during Gag assembly at the PM. To investigate the possible interaction of NC with lipid membranes in the absence of any interference from the other domains of Gag, we quantitatively characterized by fluorescence spectroscopy the binding of the mature NC protein to large unilamellar vesicles (LUVs) used as membrane models. We found that NC, either in its free form or bound to an oligonucleotide, was binding with high affinity (ϳ10 7 M ؊1 ) to negatively charged LUVs. The number of NC binding sites, but not the binding constant, was observed to decrease with the percentage of negatively charged lipids in the LUV composition, suggesting that NC and NC/oligonucleotide complexes were able to recruit negatively charged lipids to ensure optimal binding. However, in contrast to MA, NC did not exhibit a preference for phosphatidylinositol-(4,5)-bisphosphate. These results lead us to propose a modified Gag assembly model where the NC domain contributes to the initial binding of the bent form of Gag to the PM. IMPORTANCEThe NC protein is a highly conserved nucleic acid binding protein that plays numerous key roles in HIV-1 replication. While accumulating evidence shows that NC either as a mature protein or as a domain of the Gag precursor also interacts with host proteins, only a few data are available on the possible interaction of NC with lipid membranes. Interestingly, during HIV-1 assembly, the Gag precursor is thought to adopt a bent conformation where the NC domain may interact with the plasma membrane. In this context, we quantitatively characterized the binding of NC, as a free protein or as a complex with nucleic acids, to lipid membranes and showed that the latter constitute a binding platform for NC. Taken together, our data suggest that the NC domain may play a role in the initial binding events of Gag to the plasma membrane during HIV-1 assembly.T he nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) is a small basic nucleic acid binding protein with two strictly conserved CCHC motifs that strongly bind zinc (1). Mature NC results from the protease-mediated cleavage of the Gag polyprotein precursor and plays key roles in HIV-1 replication (2-5). During the early steps, NC acts as a cofactor of the reverse transcriptase (RT), to promote the initiation of reverse transcription (6-8), as well as the two obligatory strand transfers (9, 10). Moreover, NC also reduces RT pauses at the initiation step (11-13), increases the overall RT processivity (14, 15), promotes synthesis through pause sites (16-19), helps to remove the RNA fragments resulting from the RT RNase H activity (20), and may contribute to the pro...

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Section: Methodsmentioning

confidence: 55%

Section: Methodsmentioning

confidence: 99%

The HIV-1 Nucleocapsid Protein Recruits Negatively Charged Lipids To Ensure Its Optimal Binding to Lipid Membranes

Kempf

Postupalenko

Bora

et al. 2015

J Virol

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“…In this application, strategic placement of the chromophore into the macromolecule affords fluorescence changes that are directly coupled either with a displacement of the fluorophore by the analyte or to conformational changes that occur in response to analyte binding (Figure 3c). This approach has been utilized to develop sensors for various classes of analytes including carbohydrates (glucose [54–57] and maltose [58–60]), ions (nickel [61], zinc [62, 63], sulfate [64], and inorganic phosphate [65]), amino acids [66], signaling small molecules (autoinducer-2 involved in quorum sensing [67] or acyl-CoA [68]), steroids [69] and peptide-oligonucleotides [70]. …”

Section: Applicationsmentioning

confidence: 99%

Monitoring protein interactions and dynamics with solvatochromic fluorophores

Loving

Sainlos

Imperiali

2010

Trends in Biotechnology

268

196

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Solvatochromic fluorophores possess emission properties that are sensitive to the nature of the local microenvironment. These dyes have been exploited in applications ranging from the study of protein structural dynamics to the detection of protein-binding interactions. While the solvatochromic indole fluorophore of tryptophan has been utilized extensively for in vitro studies to advance our understanding of basic protein biochemistry, the emergence of new extrinsic synthetic dyes with improved properties in conjunction with recent developments in site-selective methods to incorporate these chemical tools into proteins now open the way for studies in more complex systems. Herein we discuss recent technological advancements and their application in the design of powerful reporters, which serve critical roles in modern cell biology and assay development.

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“…The 3HC probe shows a two-band emission highly sensitive to the binding of nucleic acids [31]. This two-band emission is the result of a proton transfer reaction that generates two excited states: a normal one (N*) and a tautomeric one (T*).…”

Section: Resultsmentioning

confidence: 99%

A phenyl-thiadiazolylidene-amine derivative ejects zinc from retroviral nucleocapsid zinc fingers and inactivates HIV virions

et al. 2012

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BackgroundSexual 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.ResultsHere, we have identified 2-methyl-3-phenyl-2H-[1,2,4]thiadiazol-5-ylideneamine (WDO-217) as a low-molecular-weight molecule that inactivates HIV particles. Both HIV-1 and HIV-2 virions pretreated with this compound were unable to infect permissive cells. Moreover, WDO-217 was able to inhibit infections of a wide spectrum of wild-type and drug-resistant HIV-1, including clinical isolates, HIV-2 and SIV strains. Whereas the capture of virus by DC-SIGN was unaffected by the compound, it efficiently prevented the transmission of DC-SIGN-captured virus to CD4+ T-lymphocytes. Interestingly, exposure of virions to WDO-217 reduced the amount of virion-associated genomic RNA as measured by real-time RT-qPCR. Further mechanism-of-action studies demonstrated that WDO-217 efficiently ejects zinc from the zinc fingers of the retroviral nucleocapsid protein NCp7 and inhibits the cTAR destabilization properties of this protein. Importantly, WDO-217 was able to eject zinc from both zinc fingers, even when NCp7 was bound to oligonucleotides, while no covalent interaction between NCp7 and WDO-217 could be observed.ConclusionThis compound is a new lead structure that can be used for the development of a new series of NCp7 zinc ejectors as candidate topical microbicide agents.

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Sensing peptide–oligonucleotide interactions by a two-color fluorescence label: application to the HIV-1 nucleocapsid protein

Cited by 64 publications

References 88 publications

The HIV-1 Nucleocapsid Protein Recruits Negatively Charged Lipids To Ensure Its Optimal Binding to Lipid Membranes

The HIV-1 Nucleocapsid Protein Recruits Negatively Charged Lipids To Ensure Its Optimal Binding to Lipid Membranes

Monitoring protein interactions and dynamics with solvatochromic fluorophores

A phenyl-thiadiazolylidene-amine derivative ejects zinc from retroviral nucleocapsid zinc fingers and inactivates HIV virions

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