RESUMO
The combination of distinct peptide ligation techniques to facilitate chemical protein synthesis represents one of the long-standing goals in the field. A new combination ligation method of N-to-C sequential native chemical ligation and Ser/Thr ligation (NCL-STL) is described for the first time. This method relies on the peptide salicylaldehyde S,S-propanedithioacetal (SALPDT)-ester prepared by a new 1,3-propanedithiol-mediated reaction. The peptide SALPDT-ester, which is compatible with NCL, can be fully activated by N-chlorosuccinimide (NCS)/AgNO3 in aqueous solution to afford peptide SAL-ester for use in the subsequent STL. The practicality of the combined NCL-STL method is illustrated by the synthesis of S-palmitoylated matrix-2 (S-palm M2) ion channel from Influenza A virus and S-palmitoylated interferon-induced transmembrane protein 3 (S-palm IFITM3). This approach expands the multiple-segments peptide ligation toolkit for producing important and complex custom-made protein samples by chemical protein synthesis.
Assuntos
Aldeídos/química , Ésteres/química , Proteínas de Membrana/síntese química , Propano/química , Proteínas de Ligação a RNA/síntese química , Serina/química , Compostos de Sulfidrila/química , Treonina/química , Proteínas da Matriz Viral/síntese química , Humanos , Proteínas de Membrana/química , Estrutura Molecular , Proteínas de Ligação a RNA/química , Proteínas da Matriz Viral/químicaRESUMO
Interferon-induced transmembrane proteinâ 3 (IFITM3) is an antiviral transmembrane protein that is thought to serve as the primary factor for inhibiting the replication of a large number of viruses, including West Nile virus, Dengue virus, Ebola virus, and Zika virus. Production of this 14.5â kDa, 133-residue transmembrane protein, especially with essential posttranslational modifications, by recombinant expression is challenging. In this report, we document the chemical synthesis of IFTIM3 in multi-milligram quantities (>15â mg) and the preparation of phosphorylated and fluorescent variants. The synthesis was accomplished by using KAHA ligations, which operate under acidic aqueous/organic mixtures that excel at solubilizing even the exceptionally hydrophobic C-terminal region of IFITM3. The synthetic material is readily incorporated into model vesicles and forms the basis for using synthetic, homogenous IFITM3 and its derivatives for further studying its structure and biological mode of action.
Assuntos
Proteínas de Membrana/síntese química , Proteínas de Ligação a RNA/síntese química , Sequência de Aminoácidos , Técnicas de Química Sintética/métodos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/química , Modelos Moleculares , Fosforilação , Proteínas de Ligação a RNA/químicaRESUMO
Low-complexity (LC) sequences, typically believed to be incapable of assuming structural order, are abundant constituents of the proteomes of all eukaryotic organisms. These sequences have emerged as critical components for formation of meso-scaled, sub-cellular organelles not invested by surrounding membranes, exemplified by RNA granules. We have observed that LC domains of many RNA binding proteins known to be constituents of RNA granules readily form labile cross-ß polymers under physiological conditions. Several lines of experimentation have shown that formation of labile, cross-ß polymers assembled from LC domain monomers is important for formation of RNA granules. Among the various experiments we have carried out, hydrogel binding assays have evolved as a versatile technique allowing a reliable means of assessing polymer formation and the binding of heterotypic cellular components integral to the formation of RNA granules. This article presents methods allowing for the production of hydrogel droplets composed of LC domain polymers. We further describe methods allowing straightforward assessment for binding of test LC domains to hydrogel droplets by fluorescence microscopy.
Assuntos
Hidrogéis/síntese química , Polimerização , Polímeros/síntese química , Proteínas de Ligação a RNA/síntese química , Hidrogéis/metabolismo , Polímeros/metabolismo , Ligação Proteica/fisiologia , Proteínas de Ligação a RNA/metabolismoRESUMO
The RNA recognition motif (RRM) is the largest family of eukaryotic RNA-binding proteins. Engineered RRMs with well-defined specificity would provide valuable tools and an exacting test of the current understanding of specificity. We have redesigned the specificity of an RRM using rational methods and demonstrated retargeting of its activity in cells. We engineered the conserved RRM of human Rbfox proteins to specifically bind to the terminal loop of a microRNA precursor (pre-miR-21) with high affinity and inhibit its processing by Drosha and Dicer. We further engineered Giardia Dicer by replacing its PAZ domain with the designed RRM. The reprogrammed enzyme degrades pre-miR-21 specifically in vitro and suppresses mature miR-21 levels in cells, which results in increased expression of the tumor suppressor PDCD4 and significantly decreased viability for cancer cells. The results demonstrate the feasibility of rationally engineering the sequence-specificity of RRMs and of using this ubiquitous platform for diverse biological applications.
Assuntos
MicroRNAs/biossíntese , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/farmacologia , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Moleculares , Engenharia de Proteínas , Motivo de Reconhecimento de RNA , Proteínas de Ligação a RNA/síntese química , Proteínas de Ligação a RNA/químicaRESUMO
Human telomere DNA (Htelo) and telomeric repeat-containing RNA (TERRA) are integral telomere components containing the short DNA repeats d(TTAGGG) and RNA repeats r(UUAGGG), respectively. Htelo and TERRA form G-quadruplexes, but the biological significance of their G-quadruplex formation in telomeres is unknown. Compounds that selectively bind G-quadruplex DNA and RNA are useful for understanding the functions of each G-quadruplex. Here we report that engineered Arg-Gly-Gly repeat (RGG) domains of translocated in liposarcoma containing only Phe (RGGF) and Tyr (RGGY) specifically bind and stabilize the G-quadruplexes of Htelo and TERRA, respectively. Moreover, RGGF inhibits trimethylation of both histone H4 at lysine 20 and histone H3 at lysine 9 at telomeres, while RGGY inhibits only H3 trimethylation in living cells. These findings indicate that G-quadruplexes of Htelo and TERRA have distinct functions in telomere histone methylation.
Assuntos
Quadruplex G , Engenharia de Proteínas , Proteínas de Ligação a RNA/síntese química , Arginina/química , Arginina/genética , Dicroísmo Circular , DNA/química , Ensaio de Desvio de Mobilidade Eletroforética , Glicina/química , Glicina/genética , Células HeLa , Humanos , Estrutura Terciária de Proteína/genética , RNA/química , Proteínas de Ligação a RNA/química , Sequências Repetitivas de Ácido Nucleico/genéticaRESUMO
Proteins of the pentatricopeptide repeat (PPR) superfamily are characterized by tandem arrays of a degenerate 35-amino-acid α-hairpin motif. PPR proteins are typically single-stranded RNA-binding proteins with essential roles in organelle biogenesis, RNA editing and mRNA maturation. A modular, predictable code for sequence-specific binding of RNA by PPR proteins has recently been revealed, which opens the door to the de novo design of bespoke proteins with specific RNA targets, with widespread biotechnological potential. Here, the design and production of a synthetic PPR protein based on a consensus sequence and the determination of its crystal structure to 2.2â Å resolution are described. The crystal structure displays helical disorder, resulting in electron density representing an infinite superhelical PPR protein. A structural comparison with related tetratricopeptide repeat (TPR) proteins, and with native PPR proteins, reveals key roles for conserved residues in directing the structure and function of PPR proteins. The designed proteins have high solubility and thermal stability, and can form long tracts of PPR repeats. Thus, consensus-sequence synthetic PPR proteins could provide a suitable backbone for the design of bespoke RNA-binding proteins with the potential for high specificity.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/química , Proteínas de Ligação a RNA/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Arabidopsis/síntese química , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas de Ligação a RNA/síntese química , Alinhamento de SequênciaRESUMO
The Gly- and Arg-rich C-terminal region of human nucleolin is a 61-residue long domain involved in a number of protein-protein and protein-nucleic acid interactions. This domain contains 10 aDma residues in the form of aDma-GG repeats interspersed with Phe residues. The exact role of Arg dimethylation is not known, partly because of the lack of efficient synthetic methods. This work describes an effective synthetic strategy, generally applicable to long RGG peptides, based on side-chain protected aDma and backbone protected dipeptide Fmoc-Gly-(Dmob)Gly-OH. This strategy allowed us to synthesize both the unmodified (N61Arg) and the dimethylated (N61aDma) peptides with high yield ( approximately 26%) and purity. As detected by NMR spectroscopy, N61Arg does not possess any stable secondary or tertiary structure in solution and N(omega),N(omega)-dimethylation of the guanidino group does not alter the overall conformational propensity of this peptide. While both peptides bind single-stranded nucleic acids with similar affinities (K(d) = 1.5 x 10(-7) M), they exhibit a different behaviour in ssDNA affinity chromatography consistent with the difference in pK(a) values. It has been previously shown that N61Arg inhibits HIV infection at the stage of HIV attachment to cells. This study demonstrates that Arg-dimethylated C-terminal domain lacks any inhibition activity, raising the question of whether nucleolin expressed on the cell-surface is indeed dimethylated.
Assuntos
Arginina/análogos & derivados , Fosfoproteínas/química , Proteínas de Ligação a RNA/química , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Antirretrovirais/farmacologia , Arginina/química , Cromatografia Líquida de Alta Pressão , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , HIV/efeitos dos fármacos , Células HeLa , Humanos , Espectroscopia de Ressonância Magnética , Metilação , Fosfoproteínas/síntese química , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/síntese química , NucleolinaRESUMO
A method to design novel molecules that specifically recognize a structured RNA would be a promising tool for the development of drugs or probes targeting RNA. In this study, the de novo design of the alpha-helical peptides having L-alpha-amino acids with nucleobases (nucleobase amino acids, NBAs) was carried out. Binding affinities of the peptides for a hairpin RNA derived from P22 phage were dependent on the types and positions of the NBA units they have. Some NBA peptides bound to the wild-type RNA or its mutant with high affinity and high specificity compared with the native P22 N peptide. These results indicate that the NBA units on the peptides interact with the RNA bases in a specific manner. It is demonstrated that the de novo design of peptides with the NBA units is an effective way to construct novel RNA-binding molecules.
Assuntos
Aminoácidos/química , Mutação/genética , Nucleotídeos/química , Peptídeos/síntese química , Peptídeos/metabolismo , RNA/genética , RNA/metabolismo , Dicroísmo Circular , Desenho de Fármacos , Estrutura Molecular , Conformação de Ácido Nucleico , Peptídeos/química , RNA/química , Proteínas de Ligação a RNA/síntese química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismoRESUMO
Eukaryotic aminoacyl-tRNA synthetases have dispensable extensions appended at the amino- or carboxyl-terminus as compared to their bacterial counterparts. While a synthetic peptide corresponding to the basic amino-terminal extension in yeast Asp-tRNA synthetase binds to DNA, the extension in the intact protein evidently binds to tRNA and enhances the tRNA specificity of Asp-tRNA synthetase. On the other hand, the amino-terminal extension in human Asp-tRNA synthetase, both within the intact protein and as a synthetic peptide, binds to tRNA. Here, the tRNA binding of a synthetic peptide, hKRS(Arg(25)-Glu(42)), corresponding to the amino-terminal extension of human Lys-tRNA synthetase (hKRS) was analyzed. This basic peptide bound to tRNA(Phe) and the apparent-binding constant increased with increasing concentrations of Mg(2+). The hKRS peptide also bound to DNA and polyphosphate; however, the apparent DNA-binding constants decreased at increasing concentrations of Mg(2+). The ability of the hKRS peptide to adopt alpha-helical conformation was demonstrated by NMR and circular dichroism. A Lys-rich peptide derived from the elongation factor 1alpha was also examined and bound to DNA but not to tRNA.
Assuntos
DNA/metabolismo , Lisina-tRNA Ligase/química , Peptídeos/metabolismo , RNA de Transferência/metabolismo , Sequência de Aminoácidos , Cátions Bivalentes/química , Dicroísmo Circular , DNA/química , Proteínas de Ligação a DNA/síntese química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cloreto de Magnésio/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Fator 1 de Elongação de Peptídeos/química , Peptídeos/síntese química , Peptídeos/química , Polifosfatos/química , Ligação Proteica , Estrutura Secundária de Proteína , RNA de Transferência/química , RNA de Transferência de Metionina/química , RNA de Transferência de Metionina/metabolismo , RNA de Transferência de Fenilalanina/química , RNA de Transferência de Fenilalanina/metabolismo , Proteínas de Ligação a RNA/síntese química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Cloreto de Sódio/química , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Trifluoretanol/químicaRESUMO
Site-specific modification of proteins and peptides with reporter molecules provides a powerful research tool in chemistry and biology. We report the synthesis and application of a tyrosine analogue, N-alpha-Fmoc-3-acetyl-L-tyrosine, for selective modification of proteins. As a model system, we synthesized the human immunodeficiency virus type 1 (HIV-1) Tat peptide (amino acids 47-56) containing the arginine rich RNA-binding region and replaced the Tyr-47 with 3-acetyl-tyrosine. The acetyl-Tyr-Tat peptide was subsequently labeled with a fluorescein derivative to study RNA-protein interactions by fluorescence energy transfer experiments. Our results showed that the Tat peptide binds to the rhodamine labeled TAR RNA with a dissociation constant (KD) of 1.0 +/- 0.5 nM. This strategy of selective protein modification offers a versatile new procedure for labeling peptides of biological interest at a desired site when several nucleophilic side chains of lysine and cysteine are present. These methods would provide tools for postsynthetic peptide modification and introducing biophysical probes for structural and functional analysis of proteins.
Assuntos
Produtos do Gene tat/química , Repetição Terminal Longa de HIV , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/síntese química , Engenharia de Proteínas/métodos , Proteínas de Ligação a RNA/química , Tirosina/análogos & derivados , Meios de Contraste/química , Meios de Contraste/metabolismo , Fluoresceína/química , Fluoresceína/metabolismo , Produtos do Gene tat/síntese química , Produtos do Gene tat/genética , Produtos do Gene tat/metabolismo , Genes Reporter , Humanos , Cinética , Estrutura Molecular , Conformação de Ácido Nucleico , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , RNA/genética , RNA/metabolismo , Proteínas de Ligação a RNA/síntese química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoAssuntos
Peptídeos/química , Peptídeos/síntese química , Engenharia de Proteínas/métodos , Proteínas/metabolismo , RNA/metabolismo , Sequência de Aminoácidos , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Produtos do Gene rev/genética , Produtos do Gene rev/metabolismo , Produtos do Gene tat/genética , Produtos do Gene tat/metabolismo , Cinética , Espectroscopia de Ressonância Magnética , Modelos Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas/química , RNA/química , Proteínas de Ligação a RNA/síntese química , Proteínas de Ligação a RNA/química , Elementos de Resposta , Espectrofotometria , TemperaturaRESUMO
Peptides containing peptide nucleic acid (PNA) have been designed and synthesized to construct molecules recognizing a bulge or a loop structure of RNA. Such peptides were here designed from the HIV Rev protein that can bind the stem-loop IIB of the Rev responsive element (RRE) RNA. Variations of PNA modulated the binding affinities of the peptides to RRE IIB RNA.
Assuntos
Produtos do Gene rev/química , Produtos do Gene rev/genética , Genes env/genética , HIV-1/genética , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/farmacologia , RNA Viral/metabolismo , Sequência de Aminoácidos , Animais , Arginina/metabolismo , Sítios de Ligação , Ligação Competitiva , Dicroísmo Circular , Desenho de Fármacos , Polarização de Fluorescência , Corantes Fluorescentes , Produtos do Gene rev/síntese química , HIV-1/química , Dados de Sequência Molecular , Ácidos Nucleicos Peptídicos/síntese química , Estrutura Secundária de Proteína/efeitos dos fármacos , Proteínas de Ligação a RNA/síntese química , Proteínas de Ligação a RNA/química , Rodaminas , Proteínas Virais/síntese química , Proteínas Virais/genética , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
BACKGROUND: Rop is an RNA binding, dimeric, four-helix bundle protein with a well-defined, regular hydrophobic core ideally suited for redesign studies. A family of Rop variants in which the hydrophobic core was systematically redesigned has previously been created and characterized. RESULTS: We present a structural and thermodynamic analysis of Ala2Ile2-6, a variant of Rop with an extensively redesigned hydrophobic core. The structure of Ala2Ile2-6 reveals a completely new fold formed by a conformational "flip" of the two protomers around the dimeric interface. The free-energy profile of Ala2Ile2-6 is also very different from that of wild-type Rop. Ala2Ile2-6 has a higher melting temperature than Rop, but undergoes a slightly smaller free-energy change on unfolding. CONCLUSIONS: The structure of Ala2Ile2-6, along with molecular modeling results, demonstrate the importance of tight packing of core residues and the adoption of favorable core side chain rotamer values in determining helix-helix interactions in the four-helix bundle fold. Structural disorder at the N and C termini of Ala2Ile2-6 provides a basis for the large differences in the enthalpy and entropy of Ala2Ile2-6 folding compared with wildtype Rop.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a RNA/química , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Proteínas de Bactérias/síntese química , Proteínas de Bactérias/genética , Plasmídeos de Bacteriocinas/química , Plasmídeos de Bacteriocinas/genética , Dicroísmo Circular , Cristalização , Cristalografia por Raios X , Dimerização , Escherichia coli/química , Escherichia coli/genética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Engenharia de Proteínas , Dobramento de Proteína , Isoformas de Proteínas/síntese química , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estrutura Secundária de Proteína , Proteínas de Ligação a RNA/síntese química , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/síntese química , TermodinâmicaRESUMO
An unusual and distinguishing feature of alfalfa mosaic virus (AMV) and ilarviruses such as tobacco streak virus (TSV) is that the viral coat protein is required to activate the early stages of viral RNA replication, a phenomenon known as genome activation. AMV-TSV coat protein homology is limited; however, they are functionally interchangeable in activating virus replication. For example, TSV coat protein will activate AMV RNA replication and vice versa. Although AMV and TSV coat proteins have little obvious amino acid homology, we recently reported that they share an N-terminal RNA binding consensus sequence (Ansel-McKinney et al., EMBO J. 15:5077-5084, 1996). Here, we biochemically compare the binding of chemically synthesized peptides that include the consensus RNA binding sequence and lysine-rich (AMV) or arginine-rich (TSV) environment to 3'-terminal TSV and AMV RNA fragments. The arginine-rich TSV coat protein peptide binds viral RNA with lower affinity than the lysine-rich AMV coat protein peptides; however, the ribose moieties protected from hydroxyl radical attack by the two different peptides are localized in the same area of the predicted RNA structures. When included in an infectious inoculum, both AMV and TSV 3'-terminal RNA fragments inhibited AMV RNA replication, while variant RNAs unable to bind coat protein did not affect replication significantly. The data suggest that RNA binding and genome activation functions may reside in the consensus RNA binding sequence that is apparently unique to AMV and ilarvirus coat proteins.