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Proteins are highly dynamic biomolecules that can undergo ligand-induced
conformational changes, thus often playing a crucial role in biomolecular processes. For
an in-depth understanding of protein function, the conversion of one conformational state
into another has to be resolved over space and time. Pulsed electron-electron double
resonance spectroscopy (PELDOR/DEER) in combination with site-directed spin
labelling (SDSL) is a powerful tool for obtaining distributions of interspin distances in
proteins [1, 2]. It allows for measurements with Angstrom precision, but it cannot directly
determine the time scale and the mechanism of the conformational change. However,
coupling PELDOR with rapid freeze-quench techniques adds the time axis to the
distance distribution and thus permits studying conformational changes with temporal
resolution.
Here, we show that the combination of Microsecond Freeze-Hyperquenching (MHQ) [3]
and PELDOR resolves ligand-triggered conformational changes in proteins on the
Angstrom length and microsecond time scale. It allows taking snapshots along the
trajectory of the conformational change by rapid quenching within aging times of
82-668 μs, and it is applicable at protein amounts down to 7.5 nmol (75 μM, 100 μL) per
time point. We applied MHQ/PELDOR to the cyclic nucleotide-binding domain (CNBD)
of the MloK1 channel from Mesorhizobium loti, which undergoes a conformational
change upon binding of cyclic adenosine monophosphate (cAMP). We observed a
gradual population shift from the apo to the holo state on the microsecond time scale,
but no distinct conformational intermediates (Fig. 1a, b). [4]
Figure 1: a) Interspin distance distributions obtained at different aging times and b) the corresponding
fractions of apo and holo state. c) Free-energy profile of the ligand-induced conformational change.
Corroborated by measurements of ligand-binding kinetics and molecular dynamics (MD)
simulations, we interpret the data in terms of a dwell time distribution. The transitions
across the free-energy barriers (Fig. 1c) i.e., ligand binding and the conformational
change, are on the nanosecond time scale and thus below the time resolution of the
MHQ device. However, the dwell time of the apo state in complex with the cAMP ligand
is in the microsecond range and can be monitored by MHQ/PELDOR. [4]
Literature:
[1] A.D. Milov et al., Fiz. Tverd. Tela 1981, 23, 975-982. [2] G. Jeschke, Annu. Rev. Phys. Chem.
2012, 63, 419-446. [3] A.V. Cherepanov et al., Biochim. Biophys. Acta 2004, 1656, 1-31.
[4] T. Hett et al., J. Am. Chem. Soc. 2021, 143, 6981-6989.

Given a large input graph, a k-spanner is a sparse subgraph that preserves the shortest path distances of the original within an approximation factor of k. When this distance approximation is robust to f failing nodes or edges, the spanner is f-fault tolerant. Fault tolerant spanners and their relatives arise commonly in networking and distributed computing.

There has been a recent flurry of progress on fault tolerant spanners and their relatives, including faster construction algorithms and better tradeoffs between spanner size, error, and level of fault tolerance. We will survey this progress, spanning a sequence of 7 papers over the last 5 years. We will explain the new techniques that have enabled progress, the problems that have been solved, and the problems that remain open.

Reactive oxygen species (ROS) play a key role in systemic cell to cell signaling which is required for plant acclimation to different stresses, essential for the survival of plants. We recently developed a method to image real-time whole-plant accumulation of ROS and other systemic signals, and together with transcriptomic analysis and physiological measurements, we revealed the involvement of important signaling components in response to localized high light stress leading to systemic acquired acclimation (SAA) in Arabidopsis thaliana. The signal initiation and propagation maintenance are dependent on generation of ROS by RESPIRATORY BURST OXIDASE HOMOLOGS (RBOHs) in the apoplast and transport through the plasmodesmata, under the control of PLASMODESMATA LOCALIZED PROTEIN 1 (PDLP1) and PDLP5. Furthermore, we showed that phytochrome B acts in the same regulatory module as RBOHD and that it can regulate ROS production even if it is restricted to the cytosol. Additional proteins we discovered to function in the maintenance of the signal propagation, are aquaporin PLASMA INTRINSIC PROTEIN 2;1 (PIP2;1), that transport H2O2 across the plasma membrane and calcium channels including GLUTAMATE LIKE RECEPTORS 3.3 and 3.6 (GLR3.3 & GLR3.6), MECHANOSENSORS LIKE PROTEINS 2 and 3 (MSL2 & MSL3). Based on mutants and grafting experiments we identified the role of the ROS receptor HYDROGEN PEROXIDE INDUCED CALCIUM INCREASE 1 (HPCA1) in ROS cell to cell signal propagation, as well as the calcium signal propagation. We also reported that CALCINEURIN B-LIKE CALCIUM SENSOR 4 (CBL4), CBL4 INTERACTING PROTEIN KINASE 26 (CIPK26) and OPEN STOMATA 1 (OST1) are required for the cell-to-cell ROS signals. Altogether, screening more than 120 mutants, we shed light on the underling molecular mechanisms that coordinate the systemic cell to cell signals required for plant acclimation to stress. While most of our work focused on Arabidopsis, we were able to show the ROS auto propagation systemic signals are conserved in evolution and occur also in unicellular algae colonies, non-vascular plants and even mammalian cells. Thus, emphasizing the importance of the active process of cell-to-cell ROS signaling in communicating stress response signals between cells.