RESUMO
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.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Humanos , Feminino , Drosophila melanogaster/metabolismo , Ovário/metabolismo , Transdução de Sinais/fisiologia , Oogênese/fisiologia , Ovulação , Tecido Adiposo/metabolismo , Células Germinativas/metabolismo , Aminoácidos/metabolismo , Proteínas de Drosophila/metabolismoRESUMO
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.
Assuntos
Netropsina , Nylons , Netropsina/química , DNA/química , Oligonucleotídeos , Pirróis/química , Conformação de Ácido NucleicoRESUMO
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.
Assuntos
Nanoestruturas , Ácidos Nucleicos , Nanoestruturas/química , DNA/química , Peptídeos/químicaRESUMO
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.
Assuntos
DNA Cruciforme , DNA , Conformação Molecular , Reparo do DNARESUMO
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.
Assuntos
Aminoácidos/química , Biotina/química , Proteínas Recombinantes/química , Estreptavidina/química , Sítios de Ligação , Fenômenos Biofísicos , Cristalografia por Raios X/métodos , Fluorescência , Ligantes , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
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.
Assuntos
Biotecnologia/métodos , Ouro , Nanopartículas Metálicas/química , Nanotecnologia/métodos , Peptídeos/química , Biomineralização , Cristalografia , Escherichia coli/metabolismo , Ouro/química , Ouro/metabolismoRESUMO
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.
Assuntos
Aminoácidos/química , Sítios de Ligação de Anticorpos , Ligante de CD40/química , Fluorescência , Corantes Fluorescentes/química , Fragmentos Fab das Imunoglobulinas/química , Aminoácidos/metabolismo , Ligante de CD40/metabolismo , Cristalografia por Raios X , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Modelos Moleculares , Conformação ProteicaRESUMO
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.
Assuntos
Nanoestruturas/química , Ácidos Nucleicos/química , Peptídeos/químicaRESUMO
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.
RESUMO
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.
RESUMO
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.
Assuntos
DNA/química , Cristalografia por Raios X , Conformação de Ácido Nucleico , Oligonucleotídeos/químicaRESUMO
A compelling target for the design of electron transfer proteins with novel cofactors is to create a model for the oxygen-evolving complex, a Mn4Ca cluster, of photosystem II. A mononuclear Mn cofactor can be added to the bacterial reaction center, but the addition of multiple metal centers is constrained by the native protein architecture. Alternatively, metal centers can be incorporated into artificial proteins. Designs for the addition of dinuclear metal centers to four-helix bundles resulted in three artificial proteins with ligands for one, two, or three dinuclear metal centers able to bind Mn. The three-dimensional structure determined by X-ray crystallography of one of the Mn-proteins confirmed the design features and revealed details concerning coordination of the Mn center. Electron transfer between these artificial Mn-proteins and bacterial reaction centers was investigated using optical spectroscopy. After formation of a light-induced, charge-separated state, the experiments showed that the Mn-proteins can donate an electron to the oxidized bacteriochlorophyll dimer of modified reaction centers, with the Mn-proteins having additional metal centers being more effective at this electron transfer reaction. Modeling of the structure of the Mn-protein docked to the reaction center showed that the artificial protein likely binds on the periplasmic surface similarly to cytochrome c2, the natural secondary donor. Combining reaction centers with exogenous artificial proteins provides the opportunity to create ligands and investigate the influence of inhomogeneous protein environments on multinuclear redox-active metal centers. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.
Assuntos
Proteínas de Bactérias/química , Manganês/metabolismo , Metaloproteínas/química , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Coenzimas/química , Coenzimas/genética , Coenzimas/metabolismo , Humanos , Manganês/química , Metaloproteínas/genética , Metaloproteínas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de ProteínaRESUMO
The foundational goal of structural DNA nanotechnology-the field that uses oligonucleotides as a molecular building block for the programmable self-assembly of nanostructured systems-was to use DNA to construct three-dimensional (3D) lattices for solving macromolecular structures. The programmable nature of DNA makes it an ideal system for rationally constructing self-assembled crystals and immobilizing guest molecules in a repeating 3D array through their specific stereospatial interactions with the scaffold. In this work, we have extended a previously described motif (4 × 5) by expanding the structure to a system that links four double-helical layers; we use a central weaving oligonucleotide containing a sequence of four six-base repeats (4 × 6), forming a matrix of layers that are organized and dictated by a series of Holliday junctions. In addition, we have assembled mirror image crystals (l-DNA) with the identical sequence that are completely resistant to nucleases. Bromine and selenium derivatives were obtained for the l- and d-DNA forms, respectively, allowing phase determination for both forms and solution of the resulting structures to 3.0 and 3.05 Å resolution. Both right- and left-handed forms crystallized in the trigonal space groups with mirror image 3-fold helical screw axes P32 and P31 for each motif, respectively. The structures reveal a highly organized array of discrete and well-defined cavities that are suitable for hosting guest molecules and allow us to dictate a priori the assembly of guest-DNA conjugates with a specified crystalline hand.
Assuntos
DNA/química , Bromo/química , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Nanoestruturas/química , Nanotecnologia , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Selênio/química , EstereoisomerismoRESUMO
Structural DNA nanotechnology combines branched DNA junctions with sticky-ended cohesion to create self-assembling macromolecular architectures. One of the key goals of structural DNA nanotechnology is to construct three-dimensional (3D) crystalline lattices. Here we present a new DNA motif and a strategy that has led to the assembly of a 3D lattice. We have determined the X-ray crystal structures of two related constructs to 3.1 Å resolution using bromine-derivatized crystals. The motif we used employs a five-nucleotide repeating sequence that weaves through a series of two-turn DNA duplexes. The duplexes are tied into a layered structure that is organized and dictated by a concert of four-arm junctions; these in turn assemble into continuous arrays facilitated by sequence-specific sticky-ended cohesion. The 3D X-ray structure of these DNA crystals holds promise for the design of new structural motifs to create programmable 3D DNA lattices with atomic spatial resolution. The two arrays differ by the use of four or six repeats of the five-nucleotide units in the repeating but statistically disordered central strand. In addition, we report a 2D rhombuslike array formed from similar components.
Assuntos
Cristalografia por Raios X , DNA/química , Imageamento Tridimensional , Nanotecnologia , Motivos de Aminoácidos , Bromo/química , Cristalização , Concentração de Íons de Hidrogênio , Microscopia de Força Atômica , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleotídeos/químicaRESUMO
Proteins have evolved to carry out nearly all the work required of living organisms within complex inter- and intracellular environments. However, systematically investigating the range of interactions experienced by a protein that influence its function remains challenging. DNA nanostructures are emerging as a convenient method to arrange a broad range of guest molecules. However, flexible methods are needed for arranging proteins in more biologically relevant 3D geometries under mild conditions that preserve protein function. Here we demonstrate how peptide nucleic acid (PNA) can be used to control the assembly of cytochrome c (12.5 kDa, pI 10.5) and azurin (13.9 kDa, pI 5.7) proteins into separate 3D DNA nanocages, in a process that maintains protein function. Toehold-mediated DNA strand displacement is introduced as a method to purify PNA-protein conjugates. The PNA-proteins were assembled within 2 min at room temperature and within 4 min at 11 °C, and hybridize with even greater efficiency than PNA conjugated to a short peptide. Gel electrophoresis and steady state and time-resolved fluorescence spectroscopy were used to investigate the effect of protein surface charge on its interaction with the negatively charged DNA nanocage. These data were used to generate a model of the DNA-PNA-protein complexes that show the negatively charged azurin protein repelled away from the DNA nanocage while the positively charged cytochrome c protein remains within and closely interacts with the DNA nanocage. When conjugated to PNA and incorporated into the DNA nanocage, the cytochrome c secondary structure and catalytic activity were maintained, and its redox potential was reduced modestly by 20 mV possibly due to neutralization of some positive surface charges. This work demonstrates a flexible new approach for using 3D nucleic acid (PNA-DNA) nanostructures to control the assembly of functional proteins, and facilitates further investigation of protein interactions as well as engineer more elaborate 3D protein complexes.
Assuntos
Azurina/química , Citocromos c/química , DNA/química , Nanoestruturas/química , Ácidos Nucleicos Peptídicos/química , Temperatura , Cromatografia em Gel , Modelos Moleculares , Espectrometria de FluorescênciaRESUMO
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.
Assuntos
Proteínas de Drosophila , Células-Tronco de Oogônios , Animais , Feminino , Drosophila/metabolismo , Drosophila melanogaster/genética , Células-Tronco de Oogônios/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Ovário/metabolismo , Células Germinativas/metabolismoRESUMO
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.
RESUMO
Three-dimensional (3D) DNA nanostructures have shown great promise for various applications including molecular sensing and therapeutics. Here we report kinetic studies of DNA-mediated charge transport (CT) within a 3D DNA nanostructure framework. A tetrahedral DNA nanostructure was used to investigate the through-duplex and through-space CT of small redox molecules (methylene blue (MB) and ferrocene (Fc)) that were bound to specific positions above the surface of the gold electrode. CT rate measurements provide unambiguous evidence that the intercalative MB probe undergoes efficient mediated CT over longer distances along the duplex, whereas the nonintercalative Fc probe tunnels electrons through the space. This study sheds new light on DNA-based molecular electronics and on designing high-performance biosensor devices.
Assuntos
DNA/química , Nanoestruturas/química , Sequência de Bases , Transporte Biológico , Compostos Ferrosos/química , Metalocenos , Azul de Metileno/química , Dados de Sequência Molecular , Estrutura MolecularRESUMO
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.
Assuntos
Técnicas Biossensoriais , Glicina/análogos & derivados , Fragmentos de Imunoglobulinas , Proteínas Luminescentes , Prótons , Umbeliferonas , Cristalografia por Raios X , Corantes Fluorescentes/química , Glicina/química , Glicina/genética , Fragmentos de Imunoglobulinas/química , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Espectrometria de Fluorescência , Umbeliferonas/químicaRESUMO
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.