Gd(III)-Gd(III) Relaxation-Induced Dipolar Modulation Enhancement for In-Cell Electron Paramagnetic Resonance Distance Determination

In-cell distance determination by electron paramagnetic resonance (EPR) spectroscopy reveals essential structural information about biomacromolecules under native conditions. We demonstrate that the pulsed EPR technique RIDME (relaxation induced dipolar modulation enhancement) can be utilized for such distance determination. The performance of in-cell RIDME has been assessed at Q-band using stiff molecular rulers labeled with Gd(III)-PyMTA and microinjected into Xenopus laevis oocytes. The overtone coefficients are determined to be the same for protonated aqueous solutions and inside cells. As compared to in-cell DEER (double electron-electron resonance, also abbreviated as PELDOR), in-cell RIDME features approximately 5 times larger modulation depth and does not show artificial broadening in the distance distributions due to the effect of pseudosecular terms

Dynamic Interaction of cBid with Detergents, Liposomes and Mitochondria

The BH3-only protein Bid plays a key role in the induction of mitochondrial apoptosis, but its mechanism of action is still not completely understood. Here we studied the two main activation events of Bid: Caspase-8 cleavage and interaction with the membrane bilayer. We found a striking reversible behaviour of the dissociation-association events between the Bid fragments p15 and p7. Caspase-8 cleavage does not induce per se separation of the two Bid fragments, which remain in a stable complex resembling the full length Bid. Detergents trigger a complete dissociation, which can be fully reversed by detergent removal in a range of protein concentrations from 100 µM down to 500 nM. Incubation of cBid with cardiolipin-containing liposomes leads to partial dissociation of the complex. Only p15 (tBid) fragments are found at the membrane, while p7 shows no tendency to interact with the bilayer, but complete removal of p7 strongly increases the propensity of tBid to become membrane-associated. Despite the striking structural similarities of inactive Bid and Bax, Bid does not form oligomers and reacts differently in the presence of detergents and membranes, highlighting clear differences in the modes of action of the two proteins. The partial dissociation of cBid triggered by the membrane is suggested to depend on the strong and specific interaction between p15 and p7. The reversible disassembly and re-assembly of the cBid molecules at the membrane was as well proven by EPR using spin labeled cBid in the presence of isolated mitochondria. The observed dynamic dissociation of the two Bid fragments could allow the assistance to the pore-forming Bax to occur repeatedly and may explain the proposed “hit-and-run" mode of action of Bid at the bilayer

Temperature Determination by EPR at 275 GHz and the Detection of Temperature Jumps in Aqueous Samples

Biological and Soft Matter Physic

Simulation of multi-frequency EPR spectra for a distribution of the zero-field splitting

Biological and Soft Matter Physic

Analysis of the EPR spectra of transferrin: the importance of a zero-field splitting distribution and 4th-order terms

Biological and Soft Matter Physic

Characterization of transient rheological behavior of soft materials using ferrofluid droplets

Physical material properties, such as elasticity, viscosity, or viscoelasticity, can be characterized by using rheometers or stick-type solenoid electromagnets. In this work, we developed a magnet measurement setup based on a Helmholtz arrangement of electromagnets. While applying homogeneous magnet fields to ferrofluid droplets inside a soft material of interest, the deformations of the ellipsoidal deformed droplets were measured. Kelvin-Voigt models and corresponding analytical descriptions were used to calculate the values of viscosity and Young's modulus of materials under test. For calibration purposes of the developed setup, glycerin/water mixtures and methylcellulose/water solutions were characterized as viscous and polyacrylamide gels as elastic materials, respectively. In addition, the interfacial tensions were calculated with respect to the magnetic Bond number from the droplet deformations. For the first time, the transient rheological behavior of viscoelastic material was measured using the method of ferrofluid droplet deformation. When polyacrylamide gel with a shear modulus of 230 Pa was evacuated for less than 40 min during preparation, it showed a strong time-depending viscoelastic behavior several minutes after starting the measurements. Here, Young's modulus increased up to the value of elastic behavior, whereas the values for viscosity decreased to a baseline. The developed setup can favorably be used in future applications to investigate local and also time-dependent rheological properties of soft materials