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1.
Small ; 20(9): e2307585, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-37849034

RESUMEN

The combination of multiple orthogonal interactions enables hierarchical complexity in self-assembled nanoscale materials. Here, efficient supramolecular polymerization of DNA origami nanostructures is demonstrated using a multivalent display of small molecule host-guest interactions. Modification of DNA strands with cucurbit[7]uril (CB[7]) and its adamantane guest, yielding a supramolecular complex with an affinity of order 1010 m-1 , directs hierarchical assembly of origami monomers into 1D nanofibers. This affinity regime enables efficient polymerization; a lower-affinity ß-cyclodextrin-adamantane complex does not promote extended structures at a similar valency. Finally, the utility of the high-affinity CB[7]-adamantane interactions is exploited to enable responsive enzymatic actuation of origami nanofibers assembled using peptide linkers. This work demonstrates the power of high-affinity CB[7]-guest recognition as an orthogonal axis to drive self-assembly in DNA nanotechnology.


Asunto(s)
Adamantano , Nanofibras , Nanoestructuras , Nanotecnología , ADN
2.
J Am Chem Soc ; 145(48): 26075-26085, 2023 12 06.
Artículo en Inglés | MEDLINE | ID: mdl-37987645

RESUMEN

The structural analysis of guest molecules in rationally designed and self-assembling DNA crystals has proven an elusive goal since its conception. Oligonucleotide frameworks provide an especially attractive route toward studying DNA-binding molecules by using three-dimensional lattices with defined sequence and structure. In this work, we site-specifically position a suite of minor groove binding molecules, and solve their structures via X-ray crystallography as a proof-of-principle toward scaffolding larger guest species. Two crystal motifs were used to precisely immobilize the molecules DAPI, Hoechst, and netropsin at defined positions in the lattice, allowing us to control occupancy within the crystal. We also solved the structure of a three-ring imidazole-pyrrole-pyrrole polyamide molecule, which sequence-specifically packs in an antiparallel dimeric arrangement within the minor groove. Finally, we engineered a crystal designed to position both netropsin and the polyamide at two distinct locations within the same lattice. Our work elucidates the design principles for the spatial arrangement of functional guests within lattices and opens new potential opportunities for the use of DNA crystals to display and structurally characterize small molecules, peptides, and ultimately proteins of unknown structure.


Asunto(s)
Netropsina , Nylons , Netropsina/química , ADN/química , Oligonucleótidos , Pirroles/química , Conformación de Ácido Nucleico
3.
bioRxiv ; 2023 Oct 11.
Artículo en Inglés | MEDLINE | ID: mdl-37873139

RESUMEN

The structural analysis of guest molecules in rationally designed and self-assembling DNA crystals has proven elusive since its conception. Oligonucleotide frameworks provide an especially attractive route towards studying DNA-binding molecules by using three-dimensional lattices with defined sequence and structure. In this work, we site-specifically position a suite of minor groove binding molecules, and solve their structures via x-ray crystallography, as a proof-of-principle towards scaffolding larger guest species. Two crystal motifs were used to precisely immobilize the molecules DAPI, Hoechst, and netropsin at defined positions in the lattice, allowing us to control occupancy within the crystal. We also solved the structure of a three-ring imidazole-pyrrole-pyrrole polyamide molecule, which sequence-specifically packs in an anti-parallel dimeric arrangement within the minor groove. Finally, we engineered a crystal designed to position both netropsin and the polyamide at two distinct locations within the same lattice. Our work elucidates the design principles for the spatial arrangement of functional guests within lattices and opens new potential opportunities for the use of DNA crystals to display and structurally characterize small molecules, peptides, and ultimately proteins of unknown structure.

4.
ACS Appl Bio Mater ; 2022 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-36108278

RESUMEN

The integration of proteins with DNA nanotechnology would enable materials with diverse applications in biology, medicine, and engineering. Here, we describe a method for the incorporation of bioactive fibronectin domain proteins with DNA nanostructures using two orthogonal coiled-coil peptides. One peptide from each coiled-coil pair is attached to a DNA origami cuboid in a multivalent fashion by attaching the peptides to DNA handles. These structures can then be assembled into one-dimensional arrays through the addition of a fibronectin domain linker genetically fused with the complementary peptides to those on the origami. We validate array formation using two different self-assembly protocols and characterize the fibers by atomic force and electron microscopy. Finally, we demonstrate that surfaces coated with the protein-DNA nanofibers can serve as biomaterial substrates for fibroblast adhesion and spreading with the nanofibers showing enhanced bioactivity compared to that of the monomeric protein.

5.
ACS Nano ; 15(3): 5384-5396, 2021 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-33705654

RESUMEN

Controlling the nucleation step of a self-assembly system is essential for engineering structural complexity and dynamic behaviors. Here, we design a "frame-filling" model system that comprises one type of self-complementary DNA tile and a hosting DNA origami frame to investigate the inherent dynamics of three general nucleation modes in nucleated self-assembly: unseeded, facet, and seeded nucleation. Guided by kinetic simulation, which suggested an optimal temperature range to differentiate the individual nucleation modes, and complemented by single-molecule observations, the transition of tiles from a metastable, monomeric state to a stable, polymerized state through the three nucleation pathways was monitored by Mg2+-triggered kinetic measurements. The temperature-dependent kinetics for all three nucleation modes were correlated by a "nucleation-growth" model, which quantified the tendency of nucleation using an empirical nucleation number. Moreover, taking advantage of the temperature dependence of nucleation, tile assembly can be regulated externally by the hosting frame. An ultraviolet (UV)-responsive trigger was integrated into the frame to simultaneously control "when" and "where" nucleation started. Our results reveal the dynamic mechanisms of the distinct nucleation modes in DNA tile-based self-assembly and provide a general strategy for controlling the self-assembly process.


Asunto(s)
Nanoestructuras , ADN , Cinética , Sustancias Macromoleculares , Nanotecnología
6.
Biochemistry ; 59(37): 3401-3410, 2020 09 22.
Artículo en Inglés | MEDLINE | ID: mdl-32845612

RESUMEN

Genetically encoded fluorescent noncanonical amino acids (fNCAAs) could be used to develop novel fluorescent sensors of protein function. Previous efforts toward this goal have been limited by the lack of extensive physicochemical and structural characterizations of protein-based sensors containing fNCAAs. Here, we report the steady-state spectroscopic properties and first structural analyses of an fNCAA-containing Fab fragment of the 5c8 antibody, which binds human CD40L. A previously reported 5c8 variant in which the light chain residue IleL98 is replaced with the fNCAA l-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) exhibits a 1.7-fold increase in fluorescence upon antigen binding. Determination and comparison of the apparent pKas of 7-HCAA in the unbound and bound forms indicate that the observed increase in fluorescence is not the result of perturbations in pKa. Crystal structures of the fNCAA-containing Fab in the apo and bound forms reveal interactions between the 7-HCAA side chain and surrounding residues that are disrupted upon antigen binding. This structural characterization not only provides insight into the manner in which protein environments can modulate the fluorescence properties of 7-HCAA but also could serve as a starting point for the rational design of new fluorescent protein-based reporters of protein function.


Asunto(s)
Aminoácidos/química , Sitios de Unión de Anticuerpos , Ligando de CD40/química , Fluorescencia , Colorantes Fluorescentes/química , Fragmentos Fab de Inmunoglobulinas/química , Aminoácidos/metabolismo , Ligando de CD40/metabolismo , Cristalografía por Rayos X , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Modelos Moleculares , Conformación Proteica
7.
J Am Chem Soc ; 142(3): 1406-1416, 2020 01 22.
Artículo en Inglés | MEDLINE | ID: mdl-31820959

RESUMEN

DNA and peptides are two of the most commonly used biomolecules for building self-assembling materials, but few examples exist of hybrid nanostructures that contain both components. Here we report the modification of two peptides that comprise a coiled-coil heterodimer pair with unique DNA handles in order to link DNA origami nanostructures bearing complementary strands into micrometer-long one-dimensional arrays. We probed the effect of number of coils on self-assembly and demonstrated the formation of  structures through multiple routes: one-pot assembly, formation of dimers and trimers and an alternating copolymer of two different origami structures, and stepwise assembly of purified structures with coiled-coil conjugates. Our results demonstrate the successful merging of two distinct self-assembly modes to create hybrid bionanomaterials expected to have a range of potential applications in the future.


Asunto(s)
Nanoestructuras/química , Ácidos Nucleicos/química , Péptidos/química
8.
Angew Chem Int Ed Engl ; 57(30): 9341-9345, 2018 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-29790232

RESUMEN

A reconfigurable DNA nano-tweezer is reported that can be switched between a closed and open state with a brief pulse of UV light. In its initial state, the tweezer is held shut using a hairpin with a single-stranded poly-A loop. Also incorporated in the structure is a poly-T trigger strand bearing seven photocaged residues. Upon illumination with 365 nm light, the cages are removed and the trigger strand hybridizes to the loop, opening the tweezer and increasing the distance between its arms from 4 to 18 nm. This intramolecular process is roughly 60 times faster than adding an external trigger strand, and provides a mechanism for the rapid interconversion of DNA nanostructures with light.


Asunto(s)
ADN/química , Nanoestructuras/química , Rayos Ultravioleta , Conformación de Ácido Nucleico , Procesos Fotoquímicos
9.
J Org Chem ; 81(6): 2302-7, 2016 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-26895480

RESUMEN

Recent advances in polymerase engineering have made it possible to copy information back and forth between DNA and artificial genetic polymers composed of TNA (α-L-threofuranosyl-(3',2') nucleic acid). This property, coupled with enhanced nuclease stability relative to natural DNA and RNA, warrants further investigation into the structural and functional properties of TNA as an artificial genetic polymer for synthetic biology. Here, we report a highly optimized chemical synthesis protocol for constructing multigram quantities of TNA nucleosides that can be readily converted to nucleoside 2'-phosphoramidites or 3'-triphosphates for solid-phase and polymerase-mediated synthesis, respectively. The synthetic protocol involves 10 chemical transformations with three crystallization steps and a single chromatographic purification, which results in an overall yield of 16-23% depending on the identity of the nucleoside (A, C, G, T).


Asunto(s)
ADN Polimerasa Dirigida por ADN/química , ADN/química , Ácidos Nucleicos/química , Nucleósidos/química , Oligonucleótidos/química , Compuestos Organofosforados/química , Tetrosas/síntesis química , ADN Polimerasa Dirigida por ADN/metabolismo , Ácidos Nucleicos/síntesis química , Tetrosas/química
10.
J Am Chem Soc ; 137(12): 4014-7, 2015 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-25785966

RESUMEN

Threose nucleic acid (TNA) is an unnatural genetic polymer capable of undergoing Darwinian evolution to generate folded molecules with ligand-binding activity. This property, coupled with a nuclease-resistant backbone, makes TNA an attractive candidate for future applications in biotechnology. Previously, we have shown that an engineered form of the Archaean replicative DNA polymerase 9°N, known commercially as Therminator DNA polymerase, can copy a three-letter genetic alphabet (A,T,C) from DNA into TNA. However, our ability to transcribe four-nucleotide libraries has been limited by chain termination events that prevent the synthesis of full-length TNA products. Here, we show that chain termination is caused by tG:dG mispairing in the enzyme active site. We demonstrate that the unnatural base analogue 7-deazaguanine (7dG) will suppress tGTP misincorporation by inhibiting the formation of Hoogsteen tG:dG base pairs. DNA templates that contain 7dG in place of natural dG residues replicate with high efficiency and >99% overall fidelity. Pre-steady-state kinetic measurements indicate that the rate of tCTP incorporation is 5-fold higher opposite 7dG than dG and only slightly lower than dCTP incorporation opposite either 7dG or dG. These results provide a chemical solution to the problem of how to synthesize large, unbiased pools of TNA molecules by polymerase-mediated synthesis.


Asunto(s)
Archaea/enzimología , ADN Polimerasa Dirigida por ADN/metabolismo , Guanina/análogos & derivados , Ácidos Nucleicos/química , Tetrosas/química , Emparejamiento Base , Secuencia de Bases , Guanina/química , Guanina/metabolismo , Ácidos Nucleicos/metabolismo , Tetrosas/metabolismo
11.
Bioorg Med Chem Lett ; 24(24): 5699-5703, 2014 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-25453804

RESUMEN

CD4-gp120 interaction is the first step for HIV-1 entry into host cells. A highly conserved pocket in gp120 protein is an attractive target for developing gp120 inhibitors or novel HIV detection tools. Here we incorporate seven phenylalanine derivatives having different sizes and steric conformations into position 43 of domain 1 of CD4 (mD1.2) to explore the architecture of the 'Phe43 cavity' of HIV-1 gp120. The results show that the conserved hydrophobic pocket in gp120 tolerates a hydrophobic side chain of residue 43 of CD protein, which is 12.2 Å in length and 8.0 Å in width. This result provides useful information for developing novel gp120 inhibitors or new HIV detection tools.


Asunto(s)
Antígenos CD4/química , Antígenos CD4/metabolismo , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/metabolismo , Fenilalanina/química , Fenilalanina/metabolismo , Sitios de Unión , Humanos , Modelos Moleculares , Estructura Molecular , Conformación Proteica
12.
Biochemistry ; 52(47): 8580-9, 2013 Nov 26.
Artículo en Inglés | MEDLINE | ID: mdl-24152169

RESUMEN

In a recent study, we demonstrated that structurally compact fluorophores incorporated into the side chains of amino acids could be introduced into dihydrofolate reductase from Escherichia coli (ecDHFR) with minimal disruption of protein structure or function, even when the site of incorporation was within a folded region of the protein. The modified proteins could be employed for FRET measurements, providing sensitive monitors of changes in protein conformation. The very favorable results achieved in that study encouraged us to prepare additional fluorescent amino acids of potential utility for studying protein dynamics. Presently, we describe the synthesis and photophysical characterization of four positional isomers of biphenyl-phenylalanine, all of which were found to exhibit potentially useful fluorescent properties. All four phenylalanine derivatives were used to activate suppressor tRNA transcripts and incorporated into multiple positions of ecDHFR. All phenylalanine derivatives were incorporated with good efficiency into position 16 of ecDHFR and afforded modified proteins that consumed NADPH at rates up to about twice the rate measured for wild type. This phenomenon has been noted on a number of occasions previously and shown to be due to an increase in the off-rate of tetrahydrofolate from the enzyme, altering a step that is normally rate limiting. When introduced into sterically accessible position 49, the four phenylalanine derivatives afforded DHFRs having catalytic function comparable to wild type. The four phenylalanine derivatives were also introduced into position 115 of ecDHFR, which is known to be a folded region of the protein less tolerant of structural alteration. As anticipated, significant differences were noted in the catalytic efficiencies of the derived proteins. The ability of two of the sizable biphenyl-phenylalanine derivatives to be accommodated at position 115 with minimal perturbation of DHFR function is attributed to rotational flexibility about the biphenyl bonds.


Asunto(s)
Compuestos de Bifenilo/química , Proteínas de Escherichia coli/química , Colorantes Fluorescentes/química , Indicadores y Reactivos/química , Fenilalanina/análogos & derivados , Tetrahidrofolato Deshidrogenasa/química , Biocatálisis , Compuestos de Bifenilo/síntesis química , Fenómenos Químicos , Inhibidores Enzimáticos/farmacología , Escherichia coli/enzimología , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/síntesis química , Antagonistas del Ácido Fólico/farmacología , Indicadores y Reactivos/síntesis química , Isomerismo , Metotrexato/farmacología , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/antagonistas & inhibidores , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fenilalanina/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Trimetoprim/farmacología
13.
J Am Chem Soc ; 135(35): 12924-7, 2013 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-23941571

RESUMEN

Two fluorescent amino acids, including the novel fluorescent species 4-biphenyl-l-phenylalanine (1), have been incorporated at positions 17 and 115 of dihydrofolate reductase (DHFR) to enable a study of conformational changes associated with inhibitor binding. Unlike most studies involving fluorescently labeled proteins, the fluorophores were incorporated into the amino acid side chains, and both probes [1 and L-(7-hydroxycoumarin-4-yl)ethylglycine (2)] were smaller than fluorophores typically used for such studies. The DHFR positions were chosen as potentially useful for Förster resonance energy transfer (FRET) measurements on the basis of their estimated separation (17-18 Å) and the expected change in distance along the reaction coordinate. Also of interest was the steric accessibility of the two sites: Glu17 is on the surface of DHFR, while Ile115 is within a folded region of the protein. Modified DHFR I (1 at position 17; 2 at position 115) and DHFR II (2 at position 17; 1 at position 115) were both catalytically competent. However, DHFR II containing the potentially rotatable biphenylphenylalanine moiety at sterically encumbered position 115 was significantly more active than DHFR I. Irradiation of the modified DHFRs at 280 nm effected excitation of 1, energy transfer to 2, and emission by 2 at 450 nm. However, the energy transfer was substantially more efficient in DHFR II. The effect of inhibitor binding was also measured. Trimethoprim mediated concentration-dependent diminution of the emission observed at 450 nm for DHFR II but not for DHFR I. These findings demonstrate that amino acids containing small fluorophores can be introduced into DHFR with minimal disruption of function and in a fashion that enables sensitive monitoring of changes in DHFR conformation.


Asunto(s)
Aminoácidos/análisis , Transferencia Resonante de Energía de Fluorescencia , Tetrahidrofolato Deshidrogenasa/química , Aminoácidos/química , Fluorescencia , Modelos Moleculares , Estructura Molecular , Tetrahidrofolato Deshidrogenasa/metabolismo
14.
J Am Chem Soc ; 134(46): 18883-5, 2012 Nov 21.
Artículo en Inglés | MEDLINE | ID: mdl-23116258

RESUMEN

Because of the lack of sensitivity to small changes in distance by available FRET pairs (a constraint imposed by the dimensions of the enzyme), a DHFR containing two pyrene moieties was prepared to enable the observation of excimer formation. Pyren-1-ylalanine was introduced into DHFR positions 16 and 49 using an in vitro expression system in the presence of pyren-1-ylalanyl-tRNA(CUA). Excimer formation (λ(ex) 342 nm; λ(em) 481 nm) was observed in the modified DHFR, which retained its catalytic competence and was studied under multiple and single turnover conditions. The excimer appeared to follow a protein conformational change after the H transfer involving the relative position and orientation of the pyrene moieties and is likely associated with product dissociation.


Asunto(s)
Alanina/análogos & derivados , Tetrahidrofolato Deshidrogenasa/química , Alanina/química , Transferencia Resonante de Energía de Fluorescencia , Simulación de Dinámica Molecular , Conformación Proteica
15.
Bioorg Med Chem ; 20(8): 2679-89, 2012 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-22405920

RESUMEN

N,S-diprotected L-thiothreonine and L-allo-thiothreonine derivatives were synthesized using a novel chemical strategy, and used for esterification of the dinucleotide pdCpA. The aminoacylated dinucleotides were then employed for the preparation of activated suppressor tRNA(CUA) transcripts. Thiothreonine and allo-thiothreonine were incorporated into a predetermined position of a catalytically competent dihydrofolate reductase (DHFR) analogue lacking cysteine, and the elaborated proteins were derivatized site-specifically at the thiothreonine residue with a fluorophore.


Asunto(s)
Fosfatos de Dinucleósidos/síntesis química , Fosfatos de Dinucleósidos/metabolismo , ARN de Transferencia/metabolismo , Treonina/análogos & derivados , Treonina/química , Fosfatos de Dinucleósidos/química , Estructura Molecular , ARN de Transferencia/química , Estereoisomerismo , Aminoacilación de ARN de Transferencia
16.
Biochemistry ; 51(1): 401-15, 2012 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-22145951

RESUMEN

Ribosomally mediated protein biosynthesis is limited to α-L-amino acids. A strong bias against ß-L-amino acids precludes their incorporation into proteins in vivo and also in vitro in the presence of misacylated ß-aminoacyl-tRNAs. Nonetheless, earlier studies provide some evidence that analogues of aminoacyl-tRNAs bearing ß-amino acids can be accommodated in the ribosomal A-site. Both functional and X-ray crystallographic data make it clear that the exclusion of ß-L-amino acids as participants in protein synthesis is a consequence of the architecture of the ribosomal peptidyltransferase center (PTC). To enable the reorganization of ribosomal PTC architecture through mutagenesis of 23S rRNA, a library of modified ribosomes having modifications in two regions of the 23S rRNA (2057-2063 and 2496-2507 or 2582-2588) was prepared. A dual selection procedure was used to obtain a set of modified ribosomes able to carry out protein synthesis in the presence ß-L-amino acids and to provide evidence for the utilization of such amino acids, in addition to α-L-amino acids. ß-Puromycin, a putative mimetic for ß-aminoacyl-tRNAs, was used to select modified ribosome variants having altered PTC architectures, thus potentially enabling incorporation of ß-L-amino acids. Eight types of modified ribosomes altered within the PTC have been selected by monitoring improved sensitivity to ß-puromycin in vivo. Two of the modified ribosomes, having 2057AGCGUGA2063 and 2502UGGCAG2507 or 2502AGCCAG2507, were able to suppress UAG codons in E. coli dihydrofolate reductase (DHFR) and scorpion Opisthorcanthus madagascariensis peptide IsCT mRNAs in the presence of ß-alanyl-tRNA(CUA).


Asunto(s)
Aminoácidos/química , Puromicina Aminonucleósido/análogos & derivados , Puromicina Aminonucleósido/química , ARN Ribosómico 23S/química , Proteínas Ribosómicas/química , Aminoácidos/genética , Aminoacil-ARNt Sintetasas/química , Animales , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Mutagénesis Sitio-Dirigida , Peptidil Transferasas/química , ARN Ribosómico 23S/genética , Aminoacil-ARN de Transferencia/química , Proteínas Ribosómicas/genética , Riboswitch/genética , Escorpiones/genética , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/genética , Operón de ARNr/genética
17.
Bioorg Med Chem ; 15(16): 5424-31, 2007 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-17572091

RESUMEN

Many pathogens utilize the formation of transmembrane pores in target cells in the process of infection. A great number of pore-forming proteins, both bacterial and viral, are considered to be important virulence factors, which makes them attractive targets for the discovery of new therapeutic agents. Our research is based on the idea that compounds designed to block the pores can inhibit the action of virulence factors, and that the chances to find high affinity blocking agents increase if they have the same symmetry as the target pore. Recently, we demonstrated that derivatives of beta-cyclodextrin inhibited anthrax lethal toxin (LeTx) action by blocking the transmembrane pore formed by the protective antigen (PA) subunit of the toxin. To test the broader applicability of this approach, we sought beta-cyclodextrin derivatives capable of inhibiting the activity of Staphylococcus aureus alpha-hemolysin (alpha-HL), which is regarded as a major virulence factor playing an important role in staphylococcal infection. We identified several amino acid derivatives of beta-cyclodextrin that inhibited the activity of alpha-HL and LeTx in cell-based assays at low micromolar concentrations. One of the compounds was tested for the ability to block ion conductance through the pores formed by alpha-HL and PA in artificial lipid membranes. We anticipate that this approach can serve as the basis for a structure-directed drug discovery program to find new and effective therapeutics against various pathogens that utilize pore-forming proteins as virulence factors.


Asunto(s)
Antígenos Bacterianos/metabolismo , Bacillus anthracis/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas Hemolisinas/metabolismo , Staphylococcus aureus/metabolismo , beta-Ciclodextrinas/química , beta-Ciclodextrinas/farmacología , Animales , Electrofisiología , Eritrocitos/efectos de los fármacos , Hemólisis/efectos de los fármacos , Iones/química , Ratones , Modelos Moleculares , Estructura Molecular , Conejos , beta-Ciclodextrinas/síntesis química
18.
J Am Chem Soc ; 129(12): 3586-97, 2007 Mar 28.
Artículo en Inglés | MEDLINE | ID: mdl-17338522

RESUMEN

Glycosylation of proteins can have a dramatic effect on their physical, chemical, and biological properties. Analogues of dihydrofolate reductase and firefly luciferase containing glycosylated amino acids at single, predetermined sites have been elaborated. Misacylated suppressor tRNAs activated with glycosylated serine and tyrosine derivatives were used for suppression of the nonsense codons in a cell-free protein biosynthesizing system, thereby permitting the preparation of the desired glycosylated proteins. In this fashion, it was possible to obtain proteins containing both mono- and diglycosylated amino acids, including glycosylated serine and tyrosine moieties. For the modified firefly luciferases, the effect of these substitutions on the wavelength of the light emitted by firefly luciferase was investigated. The maximum wavelength for mutants containing peracetylated glycosylated serine derivatives at position 284 showed a red shift in the emission spectra. For mutants containing glycosylated tyrosines, the red shift was observed only when the carbohydrate moiety was fully deacetylated.


Asunto(s)
Proteínas/química , Proteínas/metabolismo , Serina/metabolismo , Tirosina/metabolismo , Animales , Escherichia coli , Ésteres/química , Luciérnagas , Glicosilación , Modelos Moleculares , Conformación de Ácido Nucleico , Estructura Terciaria de Proteína , ARN de Transferencia/química , Tetrahidrofolato Deshidrogenasa/metabolismo
19.
Biochemistry ; 45(51): 15541-51, 2006 Dec 26.
Artículo en Inglés | MEDLINE | ID: mdl-17176075

RESUMEN

While numerous biologically active peptides contain D-amino acids, the elaboration of such species is not carried out by ribosomal synthesis. In fact, the bacterial ribosome discriminates strongly against the incorporation of D-amino acids from D-aminoacyl-tRNAs. To permit the incorporation of D-amino acids into proteins using in vitro protein-synthesizing systems, a strategy has been developed to prepare modified ribosomes containing alterations within the peptidyltransferase center and helix 89 of 23S rRNA. S-30 preparations derived from colonies shown to contain ribosomes with altered 23S rRNAs were found to exhibit enhanced tolerance for D-amino acids and to permit the elaboration of proteins containing D-amino acids at predetermined sites. Five specific amino acids in Escherichia coli dihydrofolate reductase and Photinus pyralis luciferase were replaced with D-phenylalanine and D-methionine, and the specific activities of the resulting enzymes were determined.


Asunto(s)
Aminoácidos/genética , Aminoácidos/metabolismo , Proteínas de Escherichia coli/genética , Ingeniería Genética , Ribosomas/genética , Acilación , Aminoácidos/química , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/química , Mutagénesis Sitio-Dirigida , Biosíntesis de Proteínas , Estructura Terciaria de Proteína/genética , ARN Ribosómico 23S/biosíntesis , ARN Ribosómico 23S/genética , ARN de Transferencia de Metionina/biosíntesis , ARN de Transferencia de Metionina/genética , ARN de Transferencia de Fenilalanina/biosíntesis , ARN de Transferencia de Fenilalanina/genética , Ribosomas/química , Ribosomas/metabolismo
20.
Antimicrob Agents Chemother ; 50(11): 3740-53, 2006 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-16982795

RESUMEN

Recently, using structure-inspired drug design, we demonstrated that aminoalkyl derivatives of beta-cyclodextrin inhibited anthrax lethal toxin action by blocking the transmembrane pore formed by the protective antigen (PA) subunit of the toxin. In the present study, we evaluate a series of new beta-cyclodextrin derivatives with the goal of identifying potent inhibitors of anthrax toxins. Newly synthesized hepta-6-thioaminoalkyl and hepta-6-thioguanidinoalkyl derivatives of beta-cyclodextrin with alkyl spacers of various lengths were tested for the ability to inhibit cytotoxicity of lethal toxin in cells as well as to block ion conductance through PA channels reconstituted in planar bilayer lipid membranes. Most of the tested derivatives were protective against anthrax lethal toxin action at low or submicromolar concentrations. They also blocked ion conductance through PA channels at concentrations as low as 0.1 nM. The activities of the derivatives in both cell protection and channel blocking were found to depend on the length and chemical nature of the substituent groups. One of the compounds was also shown to block the edema toxin activity. It is hoped that these results will help to identify a new class of drugs for anthrax treatment, i.e., drugs that block the pathway for toxin translocation into the cytosol, the PA channel.


Asunto(s)
Toxinas Bacterianas/antagonistas & inhibidores , Ciclodextrinas/síntesis química , Ciclodextrinas/farmacología , Animales , Carbunco/inmunología , Antígenos Bacterianos/inmunología , Bacillus anthracis/inmunología , Células CHO , Línea Celular , Cricetinae , AMP Cíclico/metabolismo , Ciclodextrinas/química , Indicadores y Reactivos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Espectroscopía de Resonancia Magnética , Ratones , Modelos Moleculares , Conformación Molecular , Pruebas de Neutralización
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