3D-Printing for Analytical Ultracentrifugation

Analytical ultracentrifugation (AUC) is a classical technique of physical biochemistry providing information on size, shape, and interactions of macromolecules from the analysis of their migration in centrifugal fields while free in solution. A key mechanical element in AUC is the centerpiece, a component of the sample cell assembly that is mounted between the optical windows to allow imaging and to seal the sample solution column against high vacuum while exposed to gravitational forces in excess of 300,000 g. For sedimentation velocity it needs to be precisely sector-shaped to allow unimpeded radial macromolecular migration. During the history of AUC a great variety of centerpiece designs have been developed for different types of experiments. Here, we report that centerpieces can now be readily fabricated by 3D printing at low cost, from a variety of materials, and with customized designs. The new centerpieces can exhibit sufficient mechanical stability to withstand the gravitational forces at the highest rotor speeds and be sufficiently precise for sedimentation equilibrium and sedimentation velocity experiments. Sedimentation velocity experiments with bovine serum albumin as a reference molecule in 3D printed centerpieces with standard double-sector design result in sedimentation boundaries virtually indistinguishable from those in commercial double-sector epoxy centerpieces, with sedimentation coefficients well within the range of published values. The statistical error of the measurement is slightly above that obtained with commercial epoxy, but still below 1%. Facilitated by modern open-source design and fabrication paradigms, we believe 3D printed centerpieces and AUC accessories can spawn a variety of improvements in AUC experimental design, efficiency and resource allocation.Comment: 25 pages, 6 figure

Brownian dynamics simulation of analytical ultracentrifugation experiments

<p>Abstract</p> <p>Background</p> <p>We have devised a protocol for the Brownian dynamics simulation of an analytical ultracentrifugation experiment that allows for an accurate and efficient prediction of the time-dependent concentration profiles, <it>c</it>(<it>r, t</it>) in the ultracentrifuge cell. The procedure accounts for the back-diffusion, described as a Brownian motion that superimposes to the centrifugal drift, and considers the sector-shaped geometry of the cell and the boundaries imposed by the meniscus and bottom.</p> <p>Results</p> <p>Simulations are carried out for four molecules covering a wide range of the ratio of sedimentation and diffusion coefficients. The evaluation is done by extracting the molecular parameters that were initially employed in the simulation by analyzing the profiles with an independent tool, the well-proved SEDFIT software. The code of simulation algorithm has been parallelized in order to take advantage of current multi-core computers.</p> <p>Conclusions</p> <p>Our Brownian dynamics simulation procedure may be considered as an alternative to other predictors based in numerical solutions of the Lamm equation, and its efficiency could make it useful in the most relevant, inverse problem, which is that of extracting the molecular parameters from experimentally determined concentration profiles.</p

FolX from Pseudomonas aeruginosa is octameric in both crystal and solution

FolX encodes an epimerase that forms one step of the tetrahydrofolate biosynthetic pathway, which is of interest as it is an established target for important drugs. Here we report the crystal structure of FolX from the bacterial opportunistic pathogen Pseudomonas aeruginosa, as well as a detailed analysis of the protein in solution, using analytical ultracentrifugation (AUC) and small-angle X-ray scattering (SAXS). In combination, these techniques confirm that the protein is an octamer both in the crystal structure, and in solution

The histone H3K4 demethylase JARID1A directly interacts with haematopoietic transcription factor GATA1 in erythroid cells through its second PHD domain

Chromatin remodelling and transcription factors play important roles in lineage commitment and development through control of gene expression. Activation of selected lineage-specific genes and repression of alternative lineage-affiliated genes results in tightly regulated cell differentiation transcriptional programmes. However, the complex functional and physical interplay between transcription factors and chromatin modifying enzymes remains elusive. Recent evidence has implicated histone demethylases in normal haematopoietic differentiation as well as in malignant haematopoiesis. Here we report an interaction between H3K4 demethylase JARID1A and the haematopoietic-specific master transcription proteins SCL and GATA1 in red blood cells. Specifically, we observe a direct physical contact between GATA1 and the second PHD domain of JARID1A. This interaction has potential implications for normal and malignant haematopoiesis

Inhibition by small-molecule ligands of formation of amyloid fibrils of an immunoglobulin light chain variable domain.

Overproduction of immunoglobulin light chains leads to systemic amyloidosis, a lethal disease characterized by the formation of amyloid fibrils in patients' tissues. Excess light chains are in equilibrium between dimers and less stable monomers which can undergo irreversible aggregation to the amyloid state. The dimers therefore must disassociate into monomers prior to forming amyloid fibrils. Here we identify ligands that inhibit amyloid formation by stabilizing the Mcg light chain variable domain dimer and shifting the equilibrium away from the amyloid-prone monomer

A 6 bp Z-DNA hairpin binds two Zα domains from the human RNA editing enzyme ADAR1

AbstractThe Zα domain of the human RNA editing enzyme double-stranded RNA deaminase I (ADAR1) binds to left-handed Z-DNA with high affinity. We found by analytical ultracentrifugation and CD spectroscopy that two Zα domains bind to one d(CG)3T4(CG)3 hairpin which contains a stem of six base pairs in the Z-DNA conformation. Both wild-type Zα and a C125S mutant show a mean dissociation constant of 30 nM as measured by surface plasmon resonance and analytical ultracentrifugation. Our data suggest that short (≥6 bp) segments of Z-DNA within a gene are able to recruit two ADAR1 enzymes to that particular site

Molar mass and solution conformation of branched alpha(1 - 4), alpha(1 - 6) Glucans. Part I: Glycogens in water

Solution molar masses and conformations of glycogens from different sources (rabbit, oyster, mussel and bovine) were analysed using sedimentation velocity in the analytical ultracentrifuge, size-exclusion chromatography coupled to multi-angle laser light scattering (SEC-MALLS), size-exclusion chromatography coupled to a differential pressure viscometer and dynamic light scattering. Rabbit, oyster and mussel glycogens consisted of one population of high molar mass (weight averages ranging from 4.6 x 106 to 1.1 x 107 g/mol) as demonstrated by sedimentation velocity and SEC-MALLS, whereas bovine glycogen had a bimodal distribution of significantly lower molar mass (1.0 x 105 and 4.5 x 105 g/mol). The spherical structure of all glycogen molecules was demonstrated in the slopes of the Mark-Houwink-Kuhn-Sakurada-type power-law relations for sedimentation coefficient (s20,wo), intrinsic viscosity ([η]), radius of gyration (rg,z) and radius of hydration (rH,z), respectively, and was further supported by the � (=rg,z/rH,z) function, the fractal dimension and the Perrin function. The degree of branching was estimated to be ∼10% from the shrinking factors, g′ (=[η]branched/[η]linear) and also h (=(f/fo)branched/(f/fo)linear), respectively, where (f/fo) is the translational frictional ratio, consistent with expectation. © 2007 Elsevier Ltd. All rights reserved

Spa47 is an oligomerization-activated type three secretion system (T3SS) ATPase from \u3cem\u3eShigella flexneri\u3c/em\u3e

Gram-negative pathogens often use conserved type three secretion systems (T3SS) for virulence. The Shigella type three secretion apparatus (T3SA) penetrates the host cell membrane and provides a unidirectional conduit for injection of effectors into host cells. The protein Spa47 localizes to the base of the apparatus and is speculated to be an ATPase that provides the energy for T3SA formation and secretion. Here, we developed an expression and purification protocol, producing active Spa47 and providing the first direct evidence that Spa47 is a bona fide ATPase. Additionally, size exclusion chromatography and analytical ultracentrifugation identified multiple oligomeric species of Spa47 with the largest greater than 8 fold more active for ATP hydrolysis than the monomer. An ATPase inactive Spa47 point mutant was then engineered by targeting a conserved Lysine within the predicted Walker A motif of Spa47. Interestingly, the mutant maintained a similar oligomerization pattern as active Spa47, but was unable to restore invasion phenotype when used to complement a spa47 null S. flexneri strain. Together, these results identify Spa47 as a Shigella T3SS ATPase and suggest that its activity is linked to oligomerization, perhaps as a regulatory mechanism as seen in some related pathogens. Additionally, Spa47 catalyzed ATP hydrolysis appears to be essential for host cell invasion, providing a strong platform for additional studies dissecting its role in virulence and providing an attractive target for anti-infective agents

Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin.

Gene silencing by heterochromatin is proposed to occur in part as a result of the ability of heterochromatin protein 1 (HP1) proteins to spread across large regions of the genome, compact the underlying chromatin and recruit diverse ligands. Here we identify a new property of the human HP1α protein: the ability to form phase-separated droplets. While unmodified HP1α is soluble, either phosphorylation of its N-terminal extension or DNA binding promotes the formation of phase-separated droplets. Phosphorylation-driven phase separation can be promoted or reversed by specific HP1α ligands. Known components of heterochromatin such as nucleosomes and DNA preferentially partition into the HP1α droplets, but molecules such as the transcription factor TFIIB show no preference. Using a single-molecule DNA curtain assay, we find that both unmodified and phosphorylated HP1α induce rapid compaction of DNA strands into puncta, although with different characteristics. We show by direct protein delivery into mammalian cells that an HP1α mutant incapable of phase separation in vitro forms smaller and fewer nuclear puncta than phosphorylated HP1α. These findings suggest that heterochromatin-mediated gene silencing may occur in part through sequestration of compacted chromatin in phase-separated HP1 droplets, which are dissolved or formed by specific ligands on the basis of nuclear context