David Zbaida

Kinetics and mechanism of DNA uptake into the cell nucleus

Gene transfer to eukaryotic cells requires the uptake of exogenous DNA into the cell nucleus. Except during mitosis, molecular access to the nuclear interior is limited to passage through the nuclear pores. Here we demonstrate the nuclear uptake of extended linear DNA molecules by a combination of fluorescence microscopy and single-molecule manipulation techniques, using the latter to follow uptake kinetics of individual molecules in real time. The assays were carried out on nuclei reconstituted in vitro from extracts of Xenopus eggs, which provide both a complete complement of biochemical factors involved in nuclear protein import, and unobstructed access to the nuclear pores. We find that uptake of DNA is independent of ATP or GTP hydrolysis, but is blocked by wheat germ agglutinin. The kinetics are much slower than would be expected from hydrodynamic considerations. A fit of the data to a simple model suggests femto-Newton forces and a large friction relevant to the uptake process.

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 pliant interaction network between cargo and FG repeats.

A Cyclic Continuous Process for Converting Conglomerates into Optically Pure Enantiomers by Crystallization and Dissolution with the Assistance of ‘Tailor-made’ Polymers

Abstract An industrially feasible kinetic process for the conversion of d , l racemic mixtures that crystallize in the form of conglomerates into optically pure materials was elaborated. The method takes into consideration the presence of a single-enantiomer polymer that causes crystals that match it in chirality to lag far behind their enantiomers both in growth and in dissolution. This phase lag is exploited in a repeated cycle of growth and dissolution to collect crystals of one kind after partial growth and of the other before complete dissolution. Three major steps are involved: (a) Kinetically controlled crystallization of a racemic conglomerate in the presence of chiral polymers at temperature T 1 , modeled on the basis of the structure and morphology of the 3D crystals. The polymer inhibits the precipitation of the undesired enantiomorph, collecting the desired one by filtration; (b) Preferential dissolution of added racemic mixture of crystals of the substrate in the mother liquor enriched with the undesired enantiomer and the chiral polymer at temperature T 2 . This step takes advantage of concentration-gradients of the two enantiomers in the filtrate and the enatioselective dissolution induced by the chiral polymer; (c) The separation by filtration followed by racemization of the undesired enantiomer for additional cycles of resolution. The process is illustrated for the conversion of d , l methionine·HCl that crystallizes in the form of a conglomerate that displays twinning of enantiomorphous lamellae into the corresponding l or d Met·HCl in kilograms scale.

Direct Surface Patterning from Solutions: Localized Microchemistry Using a Focused Laser

A method for creating microscale-patterned surfaces by direct-write lithography is described. A tightly focused, low-power infrared laser beam is applied to a homogeneous precursor solution containing soluble reagents. When the laser is focused directly at a glass–solution interface, it initiates the local precipitation of a solid product that attaches firmly to the substrate. Operating the laser momentarily forms isolated spots, whereas moving the microscope stage or the laser spot draws continuous lines. The method has been demonstrated for metallic silver and gold, for oxidized copper, and for molybdenum disulfide, suggesting a broad range of suitable materials. Silver patterns were further modified by chemical reactions. Their morphology and physical properties can be altered during deposition by the use of capping agents, which may provide an onset for further functionalization.

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.

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.

Design of chiral polymers for the kinetic resolution of racemic conglomerates

Abstract The design of chiral polymers as stereospecific inhibitors for efficient kinetic resolution of conglomerates by crystallization is described. The selection of the polymers is done on the basis of the crystal structure and the morphology of the substrate. The resolutions with the polymers are better than those previously obtained with low molecular weight additives. This is illustrated here for the comparative resolutions of (R,S)-glutamic acid (Glu · HCl), (R,S)-threonine, (Thr), (R,S)-asparagine monohydrate, (Asn · H2O), and (R,S)-p-hydroxyphenylglycine-p-toluenesulfonate, (pHpgpTs). Further, (R,S)-histidine, which could not be resolved with the low molecular weight inhibitors, was successfully resolved with two polymeric reagents. The method is also applicable for molecular crystals lacking ionic and hydrogen bonds as is illustrated for the resolution of (R,S)-sec-phenethyl-3,5-dinitrobenzoate (PDNB). Finally, a simultaneous resolution of the enantiomers in a device composed of two compartments separated by a membrane is described.

Design of Stereospecific Inhibitors for Crystal Dissolution

Abstract A new class of monomeric and polymeric crystal dissolution inhibitors has been prepared taking into consideration the packing arrangements and the morphologies of organic crystals. The efficiency of these inhibitors has been demonstrated by comparative morphological studies of α-glycine and by the kinetic resolution of the racemic conglomerates of his·HCl·H2O, threonine, glu·HCl, and 2,4-sec-phenethyl-3,5-dinitrobenzoate

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.

938.1-Pos Board # 190.1 – In Vitro and In Vivo Motilities of Nuclear Transport Cargos

Nuclear import of proteins involves recognition of the cargo by two helper proteins, leading to the formation of a complex, which is then translocated to the nucleus. The directionality of transport is due at least in part to the small protein Ran, present in the Ran-GTP form in the nucleus and in the hydrolyzed Ran-GDP form in the cytoplasm, and able to dissociate the cargo only in the first case. Using fluorescence correlation spectroscopy, we tested the efficiency of this molecular switch by measuring the diffusion coefficient of a fluorescent cargo in presence of the two helper proteins and of increasing concentrations of either Ran-GTP or Ran-GDP. As expected we observe an increase in the cargo mobility (signature of the complex dissociation) when Ran-GTP is added, and no change when Ran-GDP is added. We then measured the mobility of a fluorescent cargo in vivo. Whereas in the nucleus the observed mobility corresponds to the expected slightly hindered diffusion of the cargo, in the cytoplasm it is too small to correspond to the diffusion of the complex. This result could be explained either by the presence of a typical mesh size within the cytoplasm, critically slowing down the complex compared to the simple cargo, or by specific interaction of the complex with cellular structures such as the microtubule network.