Mapping conformational heterogeneity of mitochondrial nucleotide transporter in uninhibited states

One of the less understood aspects of membrane transporters is the dynamic coupling between conformational change and substrate transport, because the dynamic uninhibited states of transporters have been largely inaccessible for structural studies. Here, we use NMR approaches to investigate conformational heterogeneity of the GTP/GDP carrier (GGC) from yeast mitochondria. NMR residual dipolar coupling (RDC) analysis of GGC in DNA-origami nanotube liquid crystal shows that several structured segments have different generalized degree of order (GDO), indicating the presence of conformational heterogeneity. Complete GDO mapping reveals asymmetry between domains of the transporter and even within certain transmembrane helices. Substrate titration measurements show that nucleotide binding partially reduces local structural heterogeneity, and that the substrate binds to multiple sites along the transport cavity. Our observations suggest that mitochondrial carriers in the uninhibited states are intrinsically plastic and structural plasticity is asymmetrically distributed among the three homologous domains

Regulation at a distance of biomolecular interactions using a DNA origami nanoactuator

The creation of nanometre-sized structures that exhibit controllable motions and functions is a critical step towards building nanomachines. Recent developments in the field of DNA nanotechnology have begun to address these goals, demonstrating complex static or dynamic nanostructures made of DNA. Here we have designed and constructed a rhombus-shaped DNA origami ‘nanoactuator' that uses mechanical linkages to copy distance changes induced on one half (‘the driver') to be propagated to the other half (‘the mirror'). By combining this nanoactuator with split enhanced green fluorescent protein (eGFP), we have constructed a DNA–protein hybrid nanostructure that demonstrates tunable fluorescent behaviours via long-range allosteric regulation. In addition, the nanoactuator can be used as a sensor that responds to specific stimuli, including changes in buffer composition and the presence of restriction enzymes or specific nucleic acids

Modular Imaging Scaffold for Single-Particle Electron Microscopy

International audienceTechnological breakthroughs in electron microscopy (EM) have made it possible to solve structures of biological macromolecular complexes and to raise novel challenges, specifically related to sample preparation and heterogeneous macromolecular assemblies such as DNA–protein, protein–protein, and membrane protein assemblies. Here, we built a V-shaped DNA origami as a scaffolding molecular system to template proteins at user-defined positions in space. This template positions macromolecular assemblies of various sizes, juxtaposes combinations of biomolecules into complex arrangements, isolates biomolecules in their active state, and stabilizes membrane proteins in solution. In addition, the design can be engineered to tune DNA mechanical properties by exerting a controlled piconewton (pN) force on the molecular system and thus adapted to characterize mechanosensitive proteins. The binding site can also be specifically customized to accommodate the protein of interest, either interacting spontaneously with DNA or through directed chemical conjugation, increasing the range of potential targets for single-particle EM investigation. We assessed the applicability for five different proteins. Finally, as a proof of principle, we used RNAP protein to validate the approach and to explore the compatibility of the template with cryo-EM sample preparation

Programmable self-assembly of three-dimensional nanostructures from 104 unique components

Nucleic acids (DNA and RNA) are widely used to construct nanoscale structures with ever increasing complexity1–14 for possible applications in fields as diverse as structural biology, biophysics, synthetic biology and photonics. The nanostructures are formed through one-pot self-assembly, with early examples typically containing on the order of 10 unique DNA strands. The introduction of DNA origami4, which uses many staple strands to fold one long scaffold strand into a desired structure, gave access to kilo- to mega-dalton nanostructures containing about 102 unique DNA strands6,7,10,13 . Aiming for even larger DNA origami structures is in principle possible15,16, but faces the challenge of having to manufacture and route an increasingly long scaffold strand. An alternative and in principle more readily scalable approach uses DNA brick assembly8,9, which doesn’t need a scaffold and instead uses hundreds of short DNA brick strands that self-assemble according to specific inter-brick interactions. First-generation bricks used to create 3D structures are 32-nt long with four 8-nt binding domains that directed 102 distinct bricks into well-formed assemblies, but attempts to create larger structures encountered practical challenges and had limited success.9 Here we show that a new generation of DNA bricks with longer binding domains makes it possible to self-assemble 0.1 – 1 giga-dalton three-dimensional nanostructures from 104 unique components, including a 0.5 giga-dalton cuboid containing 30,000 unique bricks and a 1 giga-dalton rotationally symmetric tetramer. We also assemble a cuboid containing 10,000 bricks and 20,000 uniquely addressable ‘nano-voxels’ that serves as a molecular canvas for three-dimensional sculpting, with introduction of sophisticated user-prescribed 3D cavities yielding structures such as letters, a complex helicoid and a teddy bear. We anticipate that, with further optimization, even larger assemblies might be accessible and prove useful as scaffolds or for positioning functional components

Programmable self-assembly of three-dimensional nanostructures from 10,000 unique components

International audienceNucleic acids (DNA and RNA) are widely used to construct nanometre-scale structures with ever increasing complexity, with possible application in fields such as structural biology, biophysics, synthetic biology and photonics. The nanostructures are formed through one-pot self-assembly, with early kilodalton-scale examples containing typically tens of unique DNA strands. The introduction of DNA origami, which uses many staple strands to fold one long scaffold strand into a desired structure, has provided access to megadalton-scale nanostructures that contain hundreds of unique DNA strands. Even larger DNA origami structures are possible, but manufacturing and manipulating an increasingly long scaffold strand remains a challenge. An alternative and more readily scalable approach involves the assembly of DNA bricks, which each consist of four short binding domains arranged so that the bricks can interlock. This approach does not require a scaffold; instead, the short DNA brick strands self-assemble according to specific inter-brick interactions. First-generation bricks used to create three-dimensional structures are 32 nucleotides long, consisting of four eight-nucleotide binding domains. Protocols have been designed to direct the assembly of hundreds of distinct bricks into well formed structures, but attempts to create larger structures have encountered practical challenges and had limited success. Here we show that DNA bricks with longer, 13-nucleotide binding domains make it possible to self-assemble 0.1-1-gigadalton, three-dimensional nanostructures from tens of thousands of unique components, including a 0.5-gigadalton cuboid containing about 30,000 unique bricks and a 1-gigadalton rotationally symmetric tetramer. We also assembled a cuboid that contains around 10,000 bricks and about 20,000 uniquely addressable, 13-base-pair 'voxels' that serves as a molecular canvas for three-dimensional sculpting. Complex, user-prescribed, three-dimensional cavities can be produced within this molecular canvas, enabling the creation of shapes such as letters, a helicoid and a teddy bear. We anticipate that with further optimization of structure design, strand synthesis and assembly procedure even larger structures could be accessible, which could be useful for applications such as positioning functional components