Project description:Protein kinase R (PKR) is an interferon-induced kinase that plays a pivotal role in the innate immunity response to viral infection. PKR is activated upon binding to double-stranded RNA (dsRNA). Our previous analysis of binding of PKR to dsRNAs ranging from 20 to 40 bp supports a dimerization model for activation in which 30 bp represents the minimal length required to bind two PKR monomers and activate PKR via autophosphorylation. These studies were complicated by the formation of protein-RNA aggregates, particularly at low salt concentrations using longer dsRNAs. Here, we have taken advantage of the enhanced sensitivity afforded using fluorescence-detected analytical ultracentrifugation to reduce the RNA concentrations from micromolar to nanomolar. Under these conditions, we are able to characterize high-affinity binding of PKR to longer dsRNAs in 75 mM NaCl. The PKR binding stoichiometries are increased at lower salt concentrations but remain lower than those previously obtained for the dsRNA binding domain. The dependence of the limiting PKR binding stoichiometries on dsRNA length does not conform to standard models for nonspecific binding and suggests that binding to longer sequences occurs via a different binding mode with a larger site size. Although dimerization plays a key role in the PKR activation mechanism, the ability of shorter dsRNAs to bind two PKR monomers is not sufficient to induce autophosphorylation. We propose that activation of PKR by longer RNAs is correlated with an alternative binding mode in which both of the dsRNA binding motifs contact the RNA, inducing PKR to dimerize via a direct interaction of the kinase domains.
Project description:RNA-protein interactions play critical roles in gene regulation, but methods to quantitatively analyze these interactions at a large scale are lacking. We have developed a high-throughput sequencing-RNA affinity profiling (HiTS-RAP) assay by adapting a high-throughput DNA sequencer to quantify the binding of fluorescently labeled protein to millions of RNAs anchored to sequenced cDNA templates. Using HiTS-RAP, we measured the affinity of mutagenized libraries of GFP-binding and NELF-E-binding aptamers to their respective targets and identified critical regions of interaction. Mutations additively affected the affinity of the NELF-E-binding aptamer, whose interaction depended mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depended primarily on secondary structure.
Project description:We have developed a non-redundant protein-RNA binding benchmark dataset derived from the available protein-RNA structures in the Protein Database Bank. It consists of 73 complexes with measured binding affinity. The experimental conditions (pH and temperature) for binding affinity measurements are also listed in our dataset. This binding affinity dataset can be used to compare and develop protein-RNA scoring functions. The predicted binding free energy of the 73 complexes from three available scoring functions for protein-RNA docking has a low correlation with the binding Gibbs free energy calculated from Kd.
Project description:Replication factor A (RPA) is a single-strand DNA binding protein involved in DNA replication, recombination and repair processes. It is composed by the subunits RPA-1, RPA-2 and RPA-3; the major DNA-binding activity resides in the subunit 1 of the heterotrimeric RPA complex. In yeast and higher eukaryotes, besides the three basic structural DNA-binding domains, the RPA-1 subunit contains an N-terminal region involved in protein-protein interactions with a fourth DNA-binding domain. Remarkably, the N-terminal extension is absent in the RPA-1 of the pathogenic protozoan Leishmania (Leishmania) amazonensis; however, the protein maintains its ability to bind ssDNA. In a recent work, we identify Leishmania (Viannia) braziliensis RPA-1 by its specific binding to the untranslated regions of the HSP70 mRNAs, suggesting that this protein might be also an RNA-binding protein.Both rLbRPA-1 purified by His-tag affinity chromatography as well as the in vitro transcribed L. braziliensis 3' HSP70-II UTR were used to perform pull down assays to asses nucleic acid binding properties. Also, homology modeling was carried out to construct the LbRPA-1 tridimensional structure to search relevant amino acid residues to bind nucleic acids.In this work, after obtaining the recombinant L. braziliensis RPA-1 protein under native conditions, competitive and non-competitive pull-down assays confirmed the single-stranded DNA binding activity of this protein and demonstrated its interaction with the 3' UTR from the HSP70-II mRNA. As expected, this protein exhibits a high affinity for ssDNA, but we have found that RPA-1 interacts also with RNA. Additionally, we carried out a structural analysis of L. braziliensis RPA-1 protein using the X-ray diffraction structure of Ustilago maydis homologous protein as a template. Our results indicate that, in spite of the evolutionary divergence between both organisms, the structure of these two RPA-1 proteins seems to be highly conserved.The LbRPA-1 protein is a ssDNA binding protein, but also it shows affinity in vitro for the HSP70 mRNA; this finding supports a possible in vivo role in the HSP70 mRNA metabolism. On the other hand, the three dimensional model of Leishmania RPA-1 serves as a starting point for both functional analysis and its exploration as a chemotherapeutic target to combat leishmaniasis.
Project description:We utilize the recent successful overexpression of recombinant Plasmodium falciparum multi-drug resistance transporter, purification and reconstitution of the protein, and a novel high affinity chloroquine analogue to probe hypothesized interaction between the transporter and quinoline drugs. Results suggest that PfMDR1 binding sites for chloroquine, mefloquine, and quinine overlap, that P. falciparum chloroquine resistance transporter has intrinsically higher affinity for chloroquine relative to P. falciparum multi-drug resistance transporter, and that there is an isoform specific competition between the two transporters for binding of quinoline antimalarial drugs.
Project description:Eighty-eight rice (Oryza sativa) cDNAs encoding rice leaf expressed protein kinases (PKs) were fused to a Tandem Affinity Purification tag (TAP-tag) and expressed in transgenic rice plants. The TAP-tagged PKs and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by tandem mass spectrometry. Forty-five TAP-tagged PKs were recovered in this study and thirteen of these were found to interact with other rice proteins with a high probability score. In vivo phosphorylated sites were found for three of the PKs. A comparison of the TAP-tagged data from a combined analysis of 129 TAP-tagged rice protein kinases with a concurrent screen using yeast two hybrid methods identified an evolutionarily new rice protein that interacts with the well conserved cell division cycle 2 (CDC2) protein complex.
Project description:A guanine nucleotide-binding protein purified from turkey erythrocytes by affinity chromatography confers both F-- and guanine nucleotide-stimulation of adenylate cyclase to membranes from CYC- cells, a mutant cell line deficient in these responses. Interaction of turkey erythrocyte membranes with beta-adrenergic agonists before affinity chromatography, which is essential for binding of the guanine nucleotide regulatory protein to the affinity matrix, was also required for recovery of F--stimulation restoring activity in the affinity eluate.
Project description:Proteome analysis of affinity-purified materials prepared from chromatin fractions of HEK293T cells expressing various constructs. HEK293T cells transiently expressing various constructs were subjected to fanChIP (fractionation-assisted chromatin immunoprecipitation) method described in our previous paper (Okuda et al. 2014 Nucleic Acids Research 42;7 p4241-4256).