David F. Sargent

Crystal structure of the nucleosome core particle at 2.8 A resolution

The X-ray crystal structure of the nucleosome core particle of chromatin shows in atomic detail how the histone protein octamer is assembled and how 146 base pairs of DNA are organized into a superhelix around it. Both histone/histone and histone/DNA interactions depend on the histone fold domains and additional, well ordered structure elements extending from this motif. Histone amino-terminal tails pass over and between the gyres of the DNA superhelix to contact neighbouring particles. The lack of uniformity between multiple histone/DNA-binding sites causes the DNA to deviate from ideal superhelix geometry.

Solvent Mediated Interactions in the Structure of the Nucleosome Core Particle at 1.9 A Resolution

Solvent binding in the nucleosome core particle containing a 147 base pair, defined-sequence DNA is characterized from the X-ray crystal structure at 1.9 A resolution. A single-base-pair increase in DNA length over that used previously results in substantially improved clarity of the electron density and accuracy for the histone protein and DNA atomic coordinates. The reduced disorder has allowed for the first time extensive modeling of water molecules and ions. Over 3000 water molecules and 18 ions have been identified. Water molecules acting as hydrogen-bond bridges between protein and DNA are approximately equal in number to the direct hydrogen bonds between these components. Bridging water molecules have a dual role in promoting histone-DNA association not only by providing further stability to direct protein-DNA interactions, but also by enabling formation of many additional interactions between more distantly related elements. Water molecules residing in the minor groove play an important role in facilitating insertion of arginine side-chains. Water structure at the interface of the histones and DNA provides a means of accommodating intrinsic DNA conformational variation, thus limiting the sequence dependency of nucleosome positioning while enhancing mobility. Monovalent anions are bound near the N termini of histone alpha-helices that are not occluded by DNA phosphate groups. Their location in proximity to the DNA phosphodiester backbone suggests that they damp the electrostatic interaction between the histone proteins and the DNA. Divalent cations are bound at specific sites in the nucleosome core particle and contribute to histone-histone and histone-DNA interparticle interactions. These interactions may be relevant to nucleosome association in arrays.

X-ray structure of a tetranucleosome and its implications for the chromatin fibre

DNA in eukaryotic chromosomes is organized in arrays of nucleosomes compacted into chromatin fibres. This higher-order structure of nucleosomes is the substrate for DNA replication, recombination, transcription and repair. Although the structure of the nucleosome core is known at near-atomic resolution, even the most fundamental information about the organization of nucleosomes in the fibre is controversial. Here we report the crystal structure of an oligonucleosome (a compact tetranucleosome) at 9 Å resolution, solved by molecular replacement using the nucleosome core structure. The structure shows that linker DNA zigzags back and forth between two stacks of nucleosome cores, which form a truncated two-start helix, and does not follow a path compatible with a one-start solenoidal helix. The length of linker DNA is most probably buffered by stretching of the DNA contained in the nucleosome cores. We have built continuous fibre models by successively stacking tetranucleosomes one on another. The resulting models are nearly fully compacted and most closely resemble the previously described crossed-linker model. They suggest that the interfaces between nucleosomes along a single helix start are polymorphic.

Structure and mechanism of the chromatin remodelling factor ISW1a

Site-specific recognition of DNA in eukaryotic organisms depends on the arrangement of nucleosomes in chromatin. In the yeast Saccharomyces cerevisiae, ISW1a and related chromatin remodelling factors are implicated in establishing the nucleosome repeat during replication and altering nucleosome position to affect gene activity. Here we have solved the crystal structures of S. cerevisiae ISW1a lacking its ATPase domain both alone and with DNA bound at resolutions of 3.25 Å and 3.60 Å, respectively, and we have visualized two different nucleosome-containing remodelling complexes using cryo-electron microscopy. The composite X-ray and electron microscopy structures combined with site-directed photocrosslinking analyses of these complexes suggest that ISW1a uses a dinucleosome substrate for chromatin remodelling. Results from a remodelling assay corroborate the dinucleosome model. We show how a chromatin remodelling factor could set the spacing between two adjacent nucleosomes acting as a ‘protein ruler’.

Capacitance and conductance as tools for the measurement of asymmetric surface potentials and energy barriers of lipid bilayer membranes

SummaryA simple method for the determination of asymmetric surface potentials in lipid bilayers is described. The method is based on the dependence of bilayer capacitance on transmembrane voltage. The capacitance is measured by rectifying the 90° component of an applied alternating current signal. A superimposed slow triangular wave results in a hysteresis-like time course of capacitance. The center of the hysteresis figure is shifted along the voltage axis by an amount equal to the difference of the dipole plus surfacecharge potentials on the two sides of the bilayer (capacitance minimization potential).Alternatively, such bilayer asymmetry was studied by using the current-voltage characteristics in the presence of nonactin as a carrier. This analysis was based on the integrated Nernst-Planck equation, assuming a trapezoidal energy barrier and equilibrium of the surface reactions.The two methods gave consistent results for the surface potentials of phosphatidyl serine membranes asymmetrically shielded with calcium. In addition, the current analysis yields the positions of the corners of the barrier, found to be set in 13% for this lipid.

Quantitative analysis of the binding of melittin to planar lipid bilayers allowing for the discrete-charge effect

The interaction of melittin with lecithin bilayers was studied using the resulting surface potentials at the bilayer/water interfaces to monitor the association. Melittin added to the aqueous phase binds strongly to the interface but remains localized on that side of the bilayer to which it is added. The analysis of the binding curves reveals the inadequacy of the Gouy-Chapman theory for the fixed-charge surface potential in describing the electrostatic potential experienced by the adsorbed molecules. Calculations based on the Stern equation, modified for a discrete charge distribution, give a good fit to the experimental data. The thermodynamic analysis revealed different binding energies, delta G(o), at 10 and 100 mM ionic strength (-7.85 and -8.26 kcal/mol, respectively). Binding saturates at an area of 650 A2 per melittin molecule. A change in the surface dipole potential corresponding to -1.1 debye/epsilon a (epsilon a = dielectric constant of the adsorption region) had to be postulated. The Debye-Hückel length for a charge bound to the membrane/solution interface was found to be about one-third smaller than in bulk solution.

Crystal structure of Escherichia coli alkanesulfonate monooxygenase SsuD.

The FMNH(2)-dependent alkanesulfonate monooxygenase SsuD catalyzes the conversion of alkanesulfonates to the corresponding aldehyde and sulfite. The enzyme allows Escherichia coli to use a wide range of alkanesulfonates as sulfur sources for growth when sulfate or cysteine are not available. The structure of SsuD was solved using the multiwavelength anomalous dispersion method from only four ordered selenium sites per asymmetric unit (one site per 20,800 Da). The final model includes 328 of 380 amino acid residues and was refined to an R-factor of 23.5% (R(free)=27.5%) at 2.3A resolution. The X-ray crystal structure of SsuD shows a homotetrameric state for the enzyme, each subunit being composed of a TIM-barrel fold enlarged by four insertion regions that contribute to intersubunit interactions. SsuD is structurally related to a bacterial luciferase and an archaeal coenzyme F(420)-dependent reductase in spite of a low level of sequence identity with these enzymes. The structural relationship is not limited to the beta-barrel region; it includes most but not all extension regions and shows distinct properties for the SsuD TIM-barrel. A likely substrate-binding site is postulated on the basis of the SsuD structure presented here, results from earlier biochemical studies, and structure relatedness to bacterial luciferase. SsuD is related to other FMNH(2)-dependent monooxygenases that show distant sequence relationship to luciferase. Thus, the structure reported here provides a model for enzymes belonging to this family and suggests that they might all fold as TIM-barrel proteins.

Noncontact manipulation using a transversely magnetized rolling robot

A type of magnetic, wireless microrobot has been designed for non-contact manipulation of micro-objects in liquids. The agent, named the RodBot, has typical dimensions of 300 μm × 60 μm × 50 μm. The RodBot is transversely magnetized and rolls around its long axis on a surface in a rotating external magnetic field. In liquid environments, the RodBot generates a rising flow in front of it and a vortex above its body. The flow and vortex are efficient for picking-up and trapping micro-objects of sizes ranging from microns to one millimeter. In viscous solutions, a RodBot can transport objects many times its own size and weight.

Comparative analysis of the methods for measurement of membrane surface potential of planar lipid bilayers

Abstract Different methods for the measurement of the membrane surface potential Φm, or changes therein (ΔΦm), are described and compared with respect to their sensitivity, generality of application and ease of use. Examples of the measurement of ΔΦm for the determination of the surface charge density of planar lipid bilayers are presented.

Glucose minisensor based on self-assembled biotinylated phospholipid membrane on a solid support and its physical properties

We have developed a glucose minisensor based on a biotinylated, supported phospholipid membrane (s-BLM). We immobilized glucose oxidase (GOX) by coupling an avidin-GOX complex to a phospholipid bilayer formed from biotinylated crude ox brain extract (COB). The bilayer was supported on the free metal tip of a Teflon-coated stainless steel wire (diameter, 0.33 mm). The determination of glucose was based on detection of enzymatically generated hydrogen peroxide at a potential of +670 mV. We found an almost linear increase of membrane current with increasing glucose concentration up to 10 mM and a saturation effect above 30 mM glucose. A lower voltage for detection of glucose was made possible by modification of the membrane by the electron carrier TCNQ. Several methods have been used to study the physical properties of native and modified s-BLM and conventional BLM. With the electrostriction method we showed that addition of avidin-GOX complex to the electrolyte in which the biotinylated s-BLM was formed resulted in a decrease of membrane capacitance and a decrease of membrane compressibility perpendicular to the bilayer surface. The capacitance relaxation method was used to determine the changes of dielectric relaxation times (reorientation of dipole moments of polar groups of individual lipid molecules or lipid clusters) following addition of avidin-GOX. Native BLM formed from COB extract (2% solution in n-decane: butanol (8:1 v/v) exhibited one relaxation time of (5 ± 1) μs. Additional relaxation components (115 ± 27 μs and 26 ± 1 μs) appeared in BLM modified by biotin. Addition of the avidin-GOX complex to the biotinylated BLM resulted in the appearance of a slow component 505 ± 16 ms. These results clearly document the interaction of the strongly immobilized enzyme with the bilayer.