Surface plasmon resonance imaging reveals multiple binding modes of Agrobacterium transformation mediator VirE2 to ssDNA.

VirE2 is the major secreted protein of Agrobacterium tumefaciens in its genetic transformation of plant hosts. It is co-expressed with a small acidic chaperone VirE1, which prevents VirE2 oligomerization. After secretion into the host cell, VirE2 serves functions similar to a viral capsid in protecting the single-stranded transferred DNA en route to the nucleus. Binding of VirE2 to ssDNA is strongly cooperative and depends moreover on protein-protein interactions. In order to isolate the protein-DNA interactions, imaging surface plasmon resonance (SPRi) studies were conducted using surface-immobilized DNA substrates of length comparable to the protein-binding footprint. Binding curves revealed an important influence of substrate rigidity with a notable preference for poly-T sequences and absence of binding to both poly-A and double-stranded DNA fragments. Dissociation at high salt concentration confirmed the electrostatic nature of the interaction. VirE1-VirE2 heterodimers also bound to ssDNA, though by a different mechanism that was insensitive to high salt. Neither VirE2 nor VirE1-VirE2 followed the Langmuir isotherm expected for reversible monomeric binding. The differences reflect the cooperative self-interactions of VirE2 that are suppressed by VirE1.

Attenuation of encrustation by self-assembled inorganic fullerene-like nanoparticles.

Ureteral stents and urethral catheters are commonly used medical devices for maintaining urinary flow. However, long-term placement (>30 days) of these devices in the urinary tracts is limited by the development of encrustation, a phenomenon that holds a prevalence of 50% within this patient population, resulting in a great deal of morbidity to the patients. Here we report the influence of surface coating of an all-silicone catheter with rhenium-doped fullerene-like molybdenum disulfide (Re:IF-MoS2) nanoparticles on the growth and attachment of in vitro encrustation stones. Scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS) and X-ray powder diffraction (XRD) analyses indicated a remarkable attenuation in encrustation occupation on the Re:IF-MoS2-coated catheter surfaces compared to neat catheters. The doped nanoparticles displayed a unique tendency to self-assemble into mosaic-like arrangements, modifying the surface to be encrustation-repellent. The mechanism of encrustation retardation on the surface coated catheters is discussed in some detail. The ramification of these results for the clogging of other body indwelling devices is briefly discussed.

Variable internal flexibility characterizes the helical capsid formed by agrobacterium VirE2 protein on single-stranded DNA.

Agrobacterium is known for gene transfer to plants. In addition to a linear ssDNA oligonucleotide, Agrobacterium tumefaciens secretes an abundant ssDNA-binding effector, VirE2. In many ways VirE2 adapts the conjugation mechanism to transform the eukaryotic host. The crystal structure of VirE2 shows two compact domains joined by a flexible linker. Bound to ssDNA, VirE2 forms an ordered solenoidal shell, or capsid known as the T-complex. Here, we present a three-dimensional reconstruction of the VirE2-ssDNA complex using cryo-electron microscopy and iterative helical real-space reconstruction. High-resolution refinement was not possible due to inherent heterogeneity in the protein structure. By a combination of computational modeling, chemical modifications, mass spectroscopy, and electron paramagnetic resonance, we found that the N-terminal domain is tightly constrained by both tangential and longitudinal links, while the C terminus is weakly constrained. The quaternary structure is thus rigidly assembled while remaining locally flexible. This flexibility may be important in accommodating substrates without sequence specificity.

Permeating the nuclear pore complex.

The extensive and multifaceted traffic between nucleus and cytoplasm is handled by a single type of macromolecular assembly called the nuclear pore complex (NPC). While being readily accessible to ions and metabolites, the NPC imposes stringent selectivity on the passage of proteins and RNA, tightly regulating their traffic between the two major cellular compartments. Here we discuss how shuttling carriers, which mediate the transport of macromolecules through NPCs, cross its permeability barrier. We also discuss the co-existence of receptor-mediated macromolecular transport with the passive diffusion of small molecules in the context of the various models suggested for the permeability barrier of the NPC. Finally, we speculate on how nuclear transport receptors negotiate the dependence of their NPC-permeating abilities on hydrophobic interactions with the necessity of avoiding these promiscuous interactions in the cytoplasm and nucleus.

Surface functionalization of WS2 fullerene-like nanoparticles.

WS(2) belongs to a family of layered metal dichalcogenide compounds that are known to form cylindrical (inorganic nanotubes-INT) and polyhedral nanostructures--onion or nested fullerene-like (IF) particles. The outermost layers of these IF nanoparticles can be peeled under shear stress, thus IF nanoparticles have been studied for their use as solid lubricants. However, the IF nanoparticles tend to agglomerate, presumably because of surface structural defects induced by elastic strain and curvature, a fact that has a deleterious effect on their tribological properties. In the present work, chemical modification of the IF-WS(2) surface with alkyl-silane molecules is reported. The surface-modified IF nanoparticles display improved dispersion in oil-based suspensions. The alkyl-silane coating reduces the IF-WS(2) nanoparticles' tendency to agglomerate and consequently improves the long-term tribological behavior of oil formulated with the IF additive.

Cargo surface hydrophobicity is sufficient to overcome the nuclear pore complex selectivity barrier.

To fulfil their function, nuclear pore complexes (NPCs) must discriminate between inert proteins and nuclear transport receptors (NTRs), admitting only the latter. This specific permeation is thought to depend on interactions between hydrophobic patches on NTRs and phenylalanine-glycine (FG) or related repeats that line the NPC. Here, we tested this premise directly by conjugating different hydrophobic amino-acid analogues to the surface of an inert protein and examining its ability to cross NPCs unassisted by NTRs. Conjugation of as few as four hydrophobic moieties was sufficient to enable passage of the protein through NPCs. Transport of the modified protein proceeded with rates comparable to those measured for the innate protein when bound to an NTR and was relatively insensitive both to the nature and density of the amino acids used to confer hydrophobicity. The latter observation suggests a non-specific, small, and plant interaction network between cargo and FG repeats.

Synthetic mimic of selective transport through the nuclear pore complex.

The nuclear pore complex is a large protein channel present universally in eukaryotic cells. It generates an essential macromolecular separation between the nucleus and cytoplasm. The transport mechanism relies on recognition of molecular cargos by receptor proteins, and on specific interaction between the receptors and the pores. We present a chemical mimic of this "receptor-mediated" transport using modified nanoporous membrane filters, polyisopropylacrylamide as the carrier molecule, or receptor, and single-stranded DNA as the cargo. We show that a complex of ssDNA and polyisopropylacrylamide diffuses faster through the modified pores than does the bare ssDNA, in spite of the larger size of the complex. The mobile polymer thus acts as a soluble receptor to usher a macromolecular cargo specifically through the pores.

A single-step photolithographic interface for cell-free gene expression and active biochips.

We have developed a biochip platform technology suitable for controlled cell-free gene expression at the micrometer scale. A new hybrid molecule, "Daisy", was designed and synthesized to form in a single step a biocompatible lithographic interface on silicon dioxide. A protocol is described for the immobilization of linear DNA molecules thousands of base pairs long on Daisy-coated surfaces with submicrometer spatial resolution and up to high densities. On-chip protein synthesis can be obtained with a dynamic range of up to four orders of magnitude and minimal nonspecific activity. En route to on-chip artificial gene circuits, a simple two-stage gene cascade was built, in which the protein synthesized at the first location diffuses to regulate the synthesis of another protein at a second location. We demonstrate the capture of proteins from crude extract onto micrometer-scale designated traps, an important step for the formation of miniaturized self-assembled protein chips. Our biochip platform can be combined with elastomeric microfluidic devices, thereby opening possibilities for isolated and confined reaction chambers and artificial cells in which the transport of products and reagents is done by diffusion and flow. The Daisy molecule and described approach enables groups not proficient in surface chemistry to construct active biochips based on cell-free gene expression.

Dissociation of nuclear import cargo complexes by the protein Ran: a fluorescence correlation spectroscopy study.

In nucleated cells, proteins designed for nuclear import form complexes with soluble nuclear transport receptors prior to translocation across the nuclear envelope. The directionality of transport is due to the asymmetric distribution of the protein Ran, which dissociates import cargo complexes only in its nuclear RanGTP form. Using fluorescence correlation spectroscopy, we have studied the stability of cargo complexes in solution in the presence and in the absence of RanGTP. We find that RanGTP has a higher affinity for the major import receptor, the importin alpha/beta heterodimer, when importin alpha does not carry a cargo, suggesting that some nuclear transport targets might be preferentially released.