3 research outputs found
Controlled Co-reconstitution of Multiple Membrane Proteins in Lipid Bilayer Nanodiscs Using DNA as a Scaffold
Nanodiscs constitute a tool for the
solubilization of membrane
proteins in a lipid bilayer, thus offering a near-native membrane
environment. Many membrane proteins interact with other membrane proteins;
however, the co-reconstitution of multiple membrane proteins in a
single nanodisc is a random process that is adversely affected by
several factors, including protein aggregation. Here, we present an
approach for the controlled co-reconstitution of multiple membrane
proteins in a single nanodisc. The temporary attachment of designated
oligonucleotides to individual membrane proteins enables the formation
of stable, detergent-solubilized membrane protein complexes by base-pairing
of complementary oligonucleotide sequences, thus facilitating the
insertion of the membrane protein complex into nanodiscs with defined
stoichiometry and composition. As a proof of principle, nanodiscs
containing a heterodimeric and heterotrimeric membrane protein complex
were reconstituted using a fluorescently labeled voltage-gated anion
channel (VDAC) as a model system
A Programmable DNA Origami Platform to Organize SNAREs for Membrane Fusion
Soluble <i>N</i>-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)
complexes are the core molecular machinery of membrane fusion, a fundamental
process that drives inter- and intracellular communication and trafficking.
One of the questions that remains controversial has been whether and
how SNAREs cooperate. Here we show the use of self-assembled DNA-nanostructure
rings to template uniform-sized small unilamellar vesicles containing
predetermined maximal number of externally facing SNAREs to study
the membrane-fusion process. We also incorporated lipid-conjugated
complementary ssDNA as tethers into vesicle and target membranes,
which enabled bypass of the rate-limiting docking step of fusion reactions
and allowed direct observation of individual membrane-fusion events
at SNARE densities as low as one pair per vesicle. With this platform,
we confirmed at the single event level that, after docking of the
templated-SUVs to supported lipid bilayers (SBL), one to two pairs
of SNAREs are sufficient to drive fast lipid mixing. Modularity and
programmability of this platform makes it readily amenable to studying
more complicated systems where auxiliary proteins are involved
A Programmable DNA Origami Platform for Organizing Intrinsically Disordered Nucleoporins within Nanopore Confinement
Nuclear
pore complexes (NPCs) form gateways that control molecular
exchange between the nucleus and the cytoplasm. They impose a diffusion
barrier to macromolecules and enable the selective transport of nuclear
transport receptors with bound cargo. The underlying mechanisms that
establish these permeability properties remain to be fully elucidated
but require unstructured nuclear pore proteins rich in Phe-Gly (FG)-repeat
domains of different types, such as FxFG and GLFG. While physical
modeling and <i>in vitro</i> approaches have provided a
framework for explaining how the FG network contributes to the barrier
and transport properties of the NPC, it remains unknown whether the
number and/or the spatial positioning of different FG-domains along
a cylindrical, ∼40 nm diameter transport channel contributes
to their collective properties and function. To begin to answer these
questions, we have used DNA origami to build a cylinder that mimics
the dimensions of the central transport channel and can house a specified
number of FG-domains at specific positions with easily tunable design
parameters, such as grafting density and topology. We find the overall
morphology of the FG-domain assemblies to be dependent on their chemical
composition, determined by the type and density of FG-repeat, and
on their architectural confinement provided by the DNA cylinder, largely
consistent with here presented molecular dynamics simulations based
on a coarse-grained polymer model. In addition, high-speed atomic
force microscopy reveals local and reversible FG-domain condensation
that transiently occludes the lumen of the DNA central channel mimics,
suggestive of how the NPC might establish its permeability properties