Determining the Effective Density and Stabilizer Layer Thickness of Sterically Stabilized Nanoparticles.

A series of model sterically stabilized diblock copolymer nanoparticles has been designed to aid the development of analytical protocols in order to determine two key parameters: the effective particle density and the steric stabilizer layer thickness. The former parameter is essential for high resolution particle size analysis based on analytical (ultra)centrifugation techniques (e.g., disk centrifuge photosedimentometry, DCP), whereas the latter parameter is of fundamental importance in determining the effectiveness of steric stabilization as a colloid stability mechanism. The diblock copolymer nanoparticles were prepared via polymerization-induced self-assembly (PISA) using RAFT aqueous emulsion polymerization: this approach affords relatively narrow particle size distributions and enables the mean particle diameter and the stabilizer layer thickness to be adjusted independently via systematic variation of the mean degree of polymerization of the hydrophobic and hydrophilic blocks, respectively. The hydrophobic core-forming block was poly(2,2,2-trifluoroethyl methacrylate) [PTFEMA], which was selected for its relatively high density. The hydrophilic stabilizer block was poly(glycerol monomethacrylate) [PGMA], which is a well-known non-ionic polymer that remains water-soluble over a wide range of temperatures. Four series of PGMA x -PTFEMA y nanoparticles were prepared (x = 28, 43, 63, and 98, y = 100-1400) and characterized via transmission electron microscopy (TEM), dynamic light scattering (DLS), and small-angle X-ray scattering (SAXS). It was found that the degree of polymerization of both the PGMA stabilizer and core-forming PTFEMA had a strong influence on the mean particle diameter, which ranged from 20 to 250 nm. Furthermore, SAXS was used to determine radii of gyration of 1.46 to 2.69 nm for the solvated PGMA stabilizer blocks. Thus, the mean effective density of these sterically stabilized particles was calculated and determined to lie between 1.19 g cm(-3) for the smaller particles and 1.41 g cm(-3) for the larger particles; these values are significantly lower than the solid-state density of PTFEMA (1.47 g cm(-3)). Since analytical centrifugation requires the density difference between the particles and the aqueous phase, determining the effective particle density is clearly vital for obtaining reliable particle size distributions. Furthermore, selected DCP data were recalculated by taking into account the inherent density distribution superimposed on the particle size distribution. Consequently, the true particle size distributions were found to be somewhat narrower than those calculated using an erroneous single density value, with smaller particles being particularly sensitive to this artifact

The nanoscale behaviour of disordered proteins confi ned within biomimetic nuclear pore complexes

The nuclear pore complex (NPC) forms a nanochannel for selective transport into and out of the cells nucleus. Notably, the transport selectivity of the NPC critically depends on an assembly of intrinsically disordered proteins with multiple phenylalanine-glycine repeats (referred to as FG nups), grafted to the inner walls of the NPC. The focus of this work is to probe the biophysical nature of this indispensable nanochannel by using mimetic systems that emulate key properties of the NPC. Using a DNA origami scaffold, it has been possible to study the configurations adopted by purifi ed FG-nups confi ned to a nanopore geometry that mimics the NPC central channel. In this thesis, atomic force microscopy (AFM) has been used extensively to compare different flavours of FG-nups and to monitor their stochastic behaviour as they form transient condensates that occlude the pore's central lumen. Rearrangements of these entanglements were observed on the timescale of seconds, suggesting that reordering occurs at little energetic cost. This supports the idea that the FG nups are sufficiently 'sticky' or 'cohesive' to form dense condensates that seal the NPC's transport barrier yet are sufficiently dynamic at the molecular scale to facilitate the transport of larger molecules. Furthermore, the dynamics of two different FG-nup (the more cohesive Nup100 and the less cohesive Nsp1) was quantifed using autocorrelation analysis and compared to analogous data acquired on native NPCs. As predicted Nup100 condensates were found to be longer lived than those formed by its less cohesive counterpart Nsp1. By contrast, no fluctuations above the background noise were observed in native NPCs. A possible explanation being due to the presence of soluble transport receptor proteins and molecules caught in transport that trap the FG-nups in given morphologies. To further probe this hypothesis additional components of the transport system have been added to the mimetic nuclear pore complexes and their binding has been visualised with single molecule resolution, both by AFM and by total internal reflection fluorescence microscopy (TIRFM). Importin was seen to bind to both Nup100 and Nsp1 whereby a systematic increase in the number of nucleoporins led to greater binding.Open Acces

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