Demeler, Borries
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- ItemSystematic noise removal from analytical ultracentrifugation data with UltraScan(Springer Link, 2023) Mortezazadeh, Saeed; Demeler, BorriesA method for removing time- and radially invariant noise from sedimentation velocity and sedimentation equilibrium experiments performed in an analytical ultracentrifuge is presented. The method averages repeat radial incident light measurements as a function of the photomultiplier response at different wavelengths to remove the majority of the time-invariant noise contributions from intensity data measurements. The results of this method are compared to traditional absorbance data generated with a buffer reference and the Beckman Optima AUC data acquisition program, and with the standard UltraScan refinement workflow. The method avoids the amplification of stochastic noise inherent in the absorbance scan subtraction traditionally employed in sedimentation velocity and equilibrium data. In addition, the collection of intensity data frees up the reference channel for additional samples, doubling the capacity of the instrument. In comparison to absorbance data, the residual mean square deviation of a fitted sedimentation velocity experiment without additional noise correction by UltraScan was improved by a factor of 4.5 when using the new method. This improvement benefits sedimentation equilibrium experiments as well as analytical buoyant density equilibrium experiments where routine time-invariant noise correction calculations cannot be performed.
- ItemInvestigation of dynamic solution interactions between NET-1 and UNC-5B by multi-wavelength analytical ultracentrifugation(Springer Link, 2023) Gabir, Haben; Gupta, Monika; Meier, Markus; Heide, Fabian; Koch, Manuel; Stetefeld, Joerg; Demeler, BorriesNET-1 is a key chemotropic ligand that signals commissural axon migration and change in direction. NET-1 and its receptor UNC-5B switch axon growth cones from attraction to repulsion. The biophysical properties of the NET-1 + UNC-5B complex have been poorly characterized. Using multi-wavelength-AUC by adding a fluorophore to UNC-5B, we were able to separate the UNC-5B sedimentation from NET-1. Using both multi-wavelength- and single-wavelength AUC, we investigated NET-1 and UNC-5B hydrodynamic parameters and complex formation. The sedimentation velocity experiments show that NET-1 exists in a monomer–dimer equilibrium. A close study of the association shows that NET-1 forms a pH-sensitive dimer that interacts in an anti-parallel orientation. UNC-5B can form equimolar NET-1 + UNC-5B heterocomplexes with both monomeric and dimeric NET-1.
- ItemDNA supercoiling-induced shapes alter minicircle hydrodynamic properties(Oxford University Press, 2023) Waszkiewicz, Radost; Ranashinghe, Maduni; Fogg, Jonathan M.; Catanese, Daniel J.; Ekiel-Jezewska, Maria L.; Lisicki, Maciej; Demeler, Borries; Zechiedrich, Lynn; Szymczak, PiotrDNA in cells is organized in negatively supercoiled loops. The resulting torsional and bending strain allows DNA to adopt a surprisingly wide variety of 3-D shapes. This interplay between negative supercoiling, looping, and shape influences how DNA is stored, replicated, transcribed, repaired, and likely every other aspect of DNA activity. To understand the consequences of negative supercoiling and curvature on the hydrodynamic properties of DNA, we submitted 336 bp and 672 bp DNA minicircles to analytical ultracentrifugation (AUC). We found that the diffusion coefficient, sedimentation coefficient, and the DNA hydrodynamic radius strongly depended on circularity, loop length, and degree of negative supercoiling. Because AUC cannot ascertain shape beyond degree of non-globularity, we applied linear elasticity theory to predict DNA shapes, and combined these with hydrodynamic calculations to interpret the AUC data, with reasonable agreement between theory and experiment. These complementary approaches, together with earlier electron cryotomography data, provide a framework for understanding and predicting the effects of supercoiling on the shape and hydrodynamic properties of DNA.
- ItemStructure and enzymatic activity of an intellectual disability-associated ornithine decarboxylase variant, G84R(ACS Publications, 2022) Zhou, X. Edward; Schultz, Chad R.; Powell, Kelly S.; Henrickson, Amy; Lamp, Jared; Brunzelle, Joseph S.; Demeler, Borries; Vega, Irving E.; Bachmann, André S.; Melcher, KarstenOrnithine decarboxylase (ODC) is a rate-limiting enzyme for the synthesis of polyamines (PAs). PAs are required for proliferation, and increased ODC activity is associated with cancer and neural over-proliferation. ODC levels and activity are therefore tightly regulated, including through the ODC-specific inhibitor, antizyme AZ1. Recently, ODC G84R has been reported as a partial loss-of-function variant that is associated with intellectual disability and seizures. However, G84 is distant from both the catalytic center and the ODC homodimerization interface. To understand how G84R modulates ODC activity, we have determined the crystal structure of ODC G84R in both the presence and the absence of the cofactor pyridoxal 5-phosphate. The structures show that the replacement of G84 by arginine leads to hydrogen bond formation of R84 with F420, the last residue of the ODC C-terminal helix, a structural element that is involved in the AZ1-mediated proteasomal degradation of ODC. In contrast, the catalytic center is essentially indistinguishable from that of wildtype ODC. We therefore reanalyzed the catalytic activity of ODC G84R and found that it is rescued when the protein is purified in the presence of a reducing agent to mimic the reducing environment of the cytoplasm. This suggests that R84 may exert its neurological effects not through reducing ODC catalytic activity but through misregulation of its AZ1-mediated proteasomal degradation.
- ItemStructure-function studies reveal ComEA contains an oligomerization domain essential for transformation in gram-positive bacteria(Springer Nature, 2022) Ahmed, Ishtiyaq; Hahn, Jeanette; Henrickson, Amy; Khaja, Faisal Tarique; Demeler, Borries; Dubnau, David; Neiditch, Matthew B.An essential step in bacterial transformation is the uptake of DNA into the periplasm, across the thick peptidoglycan cell wall of Gram-positive bacteria, or the outer membrane and thin peptidoglycan layer of Gram-negative bacteria. ComEA, a DNA-binding protein widely conserved in transformable bacteria, is required for this uptake step. Here we determine X-ray crystal structures of ComEA from two Gram-positive species, Bacillus subtilis and Geobacillus stearothermophilus, identifying a domain that is absent in Gram-negative bacteria. X-ray crystallographic, genetic, and analytical ultracentrifugation (AUC) analyses reveal that this domain drives ComEA oligomerization, which we show is required for transformation. We use multi-wavelength AUC (MW-AUC) to characterize the interaction between DNA and the ComEA DNA-binding domain. Finally, we present a model for the interaction of the ComEA DNA-binding domain with DNA, suggesting that ComEA oligomerization may provide a pulling force that drives DNA uptake across the thick cell walls of Gram-positive bacteria.