RESUMEN
From insects to humans, oogenesis is tightly linked to nutritional input, yet little is known about how whole organism physiology matches dietary changes with oocyte development. Considering that diet-induced adipose tissue dysfunction is associated with an increased risk for fertility problems, and other obesity-associated pathophysiologies, it is critical to decipher the cellular and molecular mechanisms linking adipose nutrient sensing to remote control of the ovary and other tissues. Our previous studies in Drosophila melanogaster have shown that amino acid sensing, via the amino acid response pathway and mTOR-mediated signaling function within adipocytes to control germline stem cell maintenance and ovulation, respectively. Additionally, we demonstrated that insulin/insulin-like growth factor signaling within adipocytes employs distinct effector axes, PI3K/Akt1-dependent and -independent, downstream of insulin receptor activity to mediate fat-to-ovary communication. Here, we report that the Ras/MAPK signaling axis functions in adipocytes to regulate early germline cyst survival and ovulation of mature oocytes but is not important for germline stem cell maintenance or the progression through vitellogenesis. Thus, these studies uncover the complexity of signaling pathway activity that mediates inter-organ communication.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Animales , Humanos , Femenino , Drosophila melanogaster/metabolismo , Ovario/metabolismo , Transducción de Señal/fisiología , Oogénesis/fisiología , Ovulación , Tejido Adiposo/metabolismo , Células Germinativas/metabolismo , Aminoácidos/metabolismo , Proteínas de Drosophila/metabolismoRESUMEN
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 NucleicoRESUMEN
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.
RESUMEN
Physiological status, particularly dietary input, has major impacts on the Drosophila melanogaster ovarian germline stem cell lineage. Moreover, several studies have shed light on the role that inter-organ communication plays in coordinating whole-organism responses to changes in physiology. For example, nutrient-sensing signaling pathways function within the fat body to regulate germline stem cells and their progeny in the ovary. Together with its incredible genetic and cell biological toolkits, Drosophila serves as an amenable model organism to use for uncovering molecular mechanisms that underlie physiological control of adult stem cells. In this methods chapter, we describe a general dietary manipulation paradigm, genetic manipulation of adult adipocytes, and whole-mount ovary immunofluorescence to investigate physiological control of germline stem cells.
Asunto(s)
Proteínas de Drosophila , Células Madre Oogoniales , Animales , Femenino , Drosophila/metabolismo , Drosophila melanogaster/genética , Células Madre Oogoniales/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Ovario/metabolismo , Células Germinativas/metabolismoRESUMEN
Holliday junction (HJ) is a noncanonical four-way DNA structure with a prominent role in DNA repair, recombination, and DNA nanotechnology. By rearranging its four arms, HJ can adopt either closed or open state. With enzymes typically recognizing only a single state, acquiring detailed knowledge of the rearrangement process is an important step toward fully understanding the biological function of HJs. Here, we carried out standard all-atom molecular dynamics (MD) simulations of the spontaneous opening-closing transitions, which revealed complex conformational transitions of HJs with an involvement of previously unconsidered "half-closed" intermediates. Detailed free-energy landscapes of the transitions were obtained by sophisticated enhanced sampling simulations. Because the force field overstabilizes the closed conformation of HJs, we developed a system-specific modification which for the first time allows the observation of spontaneous opening-closing HJ transitions in unbiased MD simulations and opens the possibilities for more accurate HJ computational studies of biological processes and nanomaterials.
Asunto(s)
ADN Cruciforme , ADN , Conformación Molecular , Reparación del ADNRESUMEN
Peptides and DNA are two of the most commonly used self-assembling biological molecules for the construction of nanomaterials. However, there are only a few examples that combine these two self-assembly motifs as key structural elements in a nanostructure. We report on the synthesis of a peptide-DNA conjugate that self-assembles into a stable homotrimer based on the coiled-coil motif. The hybrid peptide-DNA trimer was then used as a novel three-way junction to link together either small DNA tile nanostructures, or to close up a triangular wireframe DNA structure. The resulting nanostructures were characterized by atomic force microscopy, and compared with a scrambled, non-assembling peptide as a control. These hybrid nanostructures enable the integration of peptide motifs and potentially bio-functionality with DNA nanostructures, and open the door to novel nano-materials that have the advantages of both molecules.
Asunto(s)
Nanoestructuras , Ácidos Nucleicos , Nanoestructuras/química , ADN/química , Péptidos/químicaRESUMEN
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.
RESUMEN
The programmable synthesis of rationally engineered crystal architectures for the precise arrangement of molecular species is a foundational goal in nanotechnology, and DNA has become one of the most prominent molecules for the construction of these materials. In particular, branched DNA junctions have been used as the central building block for the assembly of 3D lattices. Here, crystallography is used to probe the effect of all 36 immobile Holliday junction sequences on self-assembling DNA crystals. Contrary to the established paradigm in the field, most junctions yield crystals, with some enhancing the resolution or resulting in unique crystal symmetries. Unexpectedly, even the sequence adjacent to the junction has a significant effect on the crystal assemblies. Six of the immobile junction sequences are completely resistant to crystallization and thus deemed "fatal," and molecular dynamics simulations reveal that these junctions invariably lack two discrete ion binding sites that are pivotal for crystal formation. The structures and dynamics detailed here could be used to inform future designs of both crystals and DNA nanostructures more broadly, and have potential implications for the molecular engineering of applied nanoelectronics, nanophotonics, and catalysis within the crystalline context.
Asunto(s)
ADN Cruciforme , Nanoestructuras , Cristalización , ADN/química , ADN Cruciforme/genética , Nanoestructuras/química , Nanotecnología , Conformación de Ácido NucleicoRESUMEN
The fluorescent non-canonical amino acid (fNCAA) L-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) contains a photoacidic 7-hydroxycoumarin (7-HC) side chain whose fluorescence properties can be tuned by its environment. In proteins, many alterations to 7-HCAA's fluorescence spectra have been reported including increases and decreases in intensity and red- and blue-shifted emission maxima. The ability to rationally design protein environments that alter 7-HCAA's fluorescence properties in predictable ways could lead to novel protein-based sensors of biological function. However, these efforts are likely limited by a lack of structural characterization of 7-HCAA-containing proteins. Here, we report the steady-state spectroscopic and x-ray crystallographic characterization of a 7-HCAA-containing antibody fragment (in the apo and antigen-bound forms) in which a substantially blue-shifted 7-HCAA emission maximum (â¼70 nm) is observed relative to the free amino acid. Our structural characterization of these proteins provides evidence that the blue shift is a consequence of the fact that excited state proton transfer (ESPT) from the 7-HC phenol has been almost completely blocked by interactions with the protein backbone. Furthermore, a direct interaction between a residue in the antigen and the fluorophore served to further block proton transfer relative to the apoprotein. The structural basis of the unprecedented blue shift in 7-HCAA emission reported here provides a framework for the development of new fluorescent protein-based sensors.
Asunto(s)
Técnicas Biosensibles , Glicina/análogos & derivados , Fragmentos de Inmunoglobulinas , Proteínas Luminiscentes , Protones , Umbeliferonas , Cristalografía por Rayos X , Colorantes Fluorescentes/química , Glicina/química , Glicina/genética , Fragmentos de Inmunoglobulinas/química , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Espectrometría de Fluorescencia , Umbeliferonas/químicaRESUMEN
In this study, we show that maltose-binding protein (MBP) is capable of facilitating stable gold nanoparticle synthesis, and a structure of MBP in the presence of gold ions was determined by X-ray crystallography. Using this high-resolution structure of gold ion bound MBP, a peptide (AT1) was selected and synthesized and was shown to also aid in the synthesis of stable gold nanoparticles under similar experimental conditions to those used for protein facilitated synthesis. This structure-based approach represents a new potential method for the selection of peptides capable of facilitating stable nanoparticle synthesis.
Asunto(s)
Biotecnología/métodos , Oro , Nanopartículas del Metal/química , Nanotecnología/métodos , Péptidos/química , Biomineralización , Cristalografía , Escherichia coli/metabolismo , Oro/química , Oro/metabolismoRESUMEN
Fluorescent noncanonical amino acids (fNCAAs) could serve as starting points for the rational design of protein-based fluorescent sensors of biological activity. However, efforts toward this goal are likely hampered by a lack of atomic-level characterization of fNCAAs within proteins. Here, we describe the spectroscopic and structural characterization of five streptavidin mutants that contain the fNCAA l-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) at sites proximal to the binding site of its substrate, biotin. Many of the mutants exhibited altered fluorescence spectra in response to biotin binding, which included both increases and decreases in fluorescence intensity as well as red- or blue-shifted emission maxima. Structural data were also obtained for three of the five mutants. The crystal structures shed light on interactions between 7-HCAA and functional groups, contributed either by the protein or by the substrate, that may be responsible for the observed changes in the 7-HCAA spectra. These data could be used in future studies aimed at the rational design of fluorescent, protein-based sensors of small molecule binding or dissociation.
Asunto(s)
Aminoácidos/química , Biotina/química , Proteínas Recombinantes/química , Estreptavidina/química , Sitios de Unión , Fenómenos Biofísicos , Cristalografía por Rayos X/métodos , Fluorescencia , Ligandos , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
The PII-like protein SbtB has been identified as a regulator of SbtA, which is one of the key bicarbonate transporters in cyanobacteria. While SbtB from Synechocystis sp. PCC 6803 has previously been shown to be a trimer, a new crystal form is reported here which crystallizes in what is thought to be a non-native tetramer in the crystal, with the C-terminus in an extended conformation. The crystal structure shows the formation of an intermolecular disulfide bond at Cys94 between SbtB monomers, which may stabilize this conformation in the crystal. This motivates the need for future studies to investigate the potential role that the oxidation and reduction of these cysteines may play in the activation and/or function of SbtB.
Asunto(s)
Proteínas Bacterianas/química , Bicarbonatos/química , Synechocystis/química , Secuencia de Aminoácidos , Proteínas de Transporte de Anión/química , Proteínas de Transporte de Anión/genética , Proteínas de Transporte de Anión/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bicarbonatos/metabolismo , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Synechocystis/metabolismoRESUMEN
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 ProteicaRESUMEN
DNA is an ideal molecule for the construction of 3D crystals with tunable properties owing to its high programmability based on canonical Watson-Crick base pairing, with crystal assembly in all three dimensions facilitated by immobile Holliday junctions and sticky end cohesion. Despite the promise of these systems, only a handful of unique crystal scaffolds have been reported. Herein, we describe a new crystal system with a repeating sequence that mediates the assembly of a 3D scaffold via a series of Holliday junctions linked together with complementary sticky ends. By using an optimized junction sequence, we could determine a high-resolution (2.7â Å) structure containing R3 crystal symmetry, with a slight subsequent improvement (2.6â Å) using a modified sticky-end sequence. The immobile Holliday junction sequence allowed us to produce crystals that provided unprecedented atomic detail. In addition, we expanded the crystal cavities by 50 % by adding an additional helical turn between junctions, and we solved the structure to 4.5â Å resolution by molecular replacement.
RESUMEN
Hybrid protein-organometallic catalysts are being explored for selective catalysis of a number of reactions, because they utilize the complementary strengths of proteins and of organometallic complex. Herein, we present an artificial hydrogenase, StrepH2, built by incorporating a biotinylated [Fe-Fe] hydrogenase organometallic mimic within streptavidin. This strategy takes advantage of the remarkable strength and specificity of biotin-streptavidin recognition, which drives quantitative incorporation of the biotinylated diironhexacarbonyl center into streptavidin, as confirmed by UV/Vis spectroscopy and X-ray crystallography. FTIR spectra of StrepH2 show characteristic peaks at shift values indicative of interactions between the catalyst and the protein scaffold. StrepH2 catalyzes proton reduction to hydrogen in aqueous media during photo- and electrocatalysis. Under photocatalytic conditions, the protein-embedded catalyst shows enhanced efficiency and prolonged activity compared to the isolated catalyst. Transient absorption spectroscopy data suggest a mechanism for the observed increase in activity underpinned by an observed longer lifetime for the catalytic species FeI Fe0 when incorporated within streptavidin compared to the biotinylated catalyst in solution.
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/químicaRESUMEN
Three-dimensional (3D) cages are one of the most important targets for nanotechnology. Both proteins and DNA have been used as building blocks to create tunable nanoscale cages for a wide range of applications, but each molecular type has its own limitations. Here, we report a cage constructed from both protein and DNA building blocks through the use of covalent protein-DNA conjugates. We modified a homotrimeric protein (KDPG aldolase) with three identical single-stranded DNA handles by functionalizing a reactive cysteine residue introduced via site-directed mutagenesis. This protein-DNA building block was coassembled with a triangular DNA structure bearing three complementary arms to the handles, resulting in tetrahedral cages comprising six DNA sides capped by the protein trimer. The dimensions of the cage could be tuned through the number of turns per DNA arm (3 turns â¼ 10 nm, 4 turns â¼ 14 nm), and the hybrid structures were purified and characterized to confirm the three-dimensional structure. Cages were also modified with DNA using click chemistry and using aldolase trimers bearing the noncanonical amino acid 4-azidophenylalanine, demonstrating the generality of the method. Our approach will allow for the construction of nanomaterials that possess the advantages of both protein and DNA nanotechnology and find applications in fields such as targeted delivery, structural biology, biomedicine, and catalytic materials.
Asunto(s)
Aldehído-Liasas/química , ADN/química , Nanoestructuras/química , Nanotecnología , Aldehído-Liasas/genética , Aldehído-Liasas/metabolismo , Humanos , Modelos MolecularesRESUMEN
Three-dimensional (3D) DNA nanostructures facilitate the directed self-assembly of various objects with designed patterns with nanometer scale addressability. Here, we report the enhancement of cytochrome c (cyt c) redox activity by using a designed 3D DNA nanostructure attached to a gold electrode to spatially control the position of cyt c within the tetrahedral framework. Charged encapsulation and spatial control result in the significantly increased redox potential and enhanced electron transfer of this redox protein when compared to cyt c directly adsorbed on the gold surface. Two different protein attachment sites on one double stranded edge of a DNA tetrahedron were used to position cyt c inside and outside of the cage. Cyt c at both binding sites show similar redox potential shift and only slight difference in the electron transfer rate, both orders of magnitude faster than the cases when the protein was directly deposited on the gold electrode, likely due to an effective electron transfer pathway provided by the stabilization effect of the protein created by the DNA framework. This study shows great potential of using structural DNA nanotechnology for spatial control of protein positioning on electrode, which opens new routes to engineer redox proteins and interface microelectronic devices with biological function.
Asunto(s)
Citocromos c/química , ADN/química , Nanoestructuras/química , Citocromos c/metabolismo , Técnicas Electroquímicas , Electrodos , Transporte de Electrón , Oro/química , Oxidación-ReducciónRESUMEN
Programming self-assembled designer DNA crystals with various lattices and functions is one of the most important goals for nanofabrication using nucleic acids. The resulting porous materials possess atomic precision for several potential applications that rely on crystalline lattices and cavities. Herein, we present a rationally designed and self-assembled 3D DNA crystal lattice with hexagonal symmetry. In our design, two 21-base oligonucleotides are used to form a duplex motif that further assembles into a 3D array. The interactions between the strands are programmed using Watson-Crick base-pairing. The six-fold symmetry, as well as the chirality, is directed by the Holliday junctions formed between the duplex motifs. The rationally designed DNA crystal provides a new avenue that could create self-assembled macromolecular 3D crystalline lattices with atomic precision. In addition, the structure contains a highly organized array of well-defined cavities that are suitable for future applications with immobilized guests.