Shuttle protein UBQLN2 functions in protein quality control (PQC) by binding to proteasomal receptors and ubiquitinated substrates via its N‐terminal ubiquitin‐like (UBL) and C‐terminal ubiquitin‐associated (UBA) domains, respectively. Between these two folded domains are low‐complexity STI1‐I and STI1‐II regions, connected by disordered linkers. The STI1 regions bind other components, such as HSP70, that are important to the PQC functions of UBQLN2. We recently determined that the STI1‐II region enables UBQLN2 to undergo liquid–liquid phase separation (LLPS) to form liquid droplets in vitro and biomolecular condensates in cells. However, how the interplay between the folded (UBL/UBA) domains and the intrinsically disordered regions mediates phase separation is largely unknown. Using engineered domain deletion constructs, we found that removing the UBA domain inhibits UBQLN2 LLPS while removing the UBL domain enhances LLPS, suggesting that UBA and UBL domains contribute asymmetrically in modulating UBQLN2 LLPS. To explain these differential effects, we interrogated the interactions that involve the UBA and UBL domains across the entire UBQLN2 molecule using nuclear magnetic resonance spectroscopy. To our surprise, aside from well‐studied canonical UBL:UBA interactions, there also exist moderate interactions between the UBL and several disordered regions, including STI1‐I and residues 555–570, the latter of which is a known contributor to UBQLN2 LLPS. Our findings are essential for the understanding of both the molecular driving forces of UBQLN2 LLPS and the effects of ligand binding to UBL, UBA, or disordered regions on the phase behavior and physiological functions of UBQLN2.

The interactions of single‐stranded DNA (ssDNA) immobilized on gold electrodes with hexammine ruthenium(III) (RuHex) and hexammine cobalt(III) (CoHex) were investigated with chronocoulometric and electrochemical quartz crystal microbalance experiments. The modified surfaces were created by immobilizing ssDNA with tris(dithioserinol) linkers to form a compact, self‐assembled monolayer, followed by passivation with 6‐mercapto‐1‐hexanol (MCH). Upon addition of the redox indicator during a potential jump for RuHex and CoHex, we observed a dramatic increase in the frequency response in comparison to the signal observed with only low‐ionic‐strength hybridization buffer, 10 mM tris(hydroxymethyl)‐aminomethane (Tris)/H2SO4 (pH 7.4). RuHex and CoHex responded quite differently, even on a millisecond time scale. As the expected mass change is less than a factor of 35 to the observed frequency response, we concluded that the applied potential jumps lead to switching of the DNA layer viscoelasticity.

We report on H/D isotope effects observed upon quick redox‐switching of the viscoelasticity of self‐assembled monolayers of single‐stranded DNA (ssDNA‐SAM) observed by electrochemical quartz‐crystal micro‐balance (EQCM) of three redox‐active small molecules that travel through the DNA layer on gold electrodes. We have recently reported hexammine cobalt(III) (CoHex) to have the largest voltammetric isotope effect while hexammine ruthenium(III) (RuHex) does not show this effect. Daunomycin, on the other hand showed a significant redox potential shift up to −80 mV. A thin‐layer model may explain this voltammetric behavior. RuHex covers the negatively charged DNA strand and provides considerable conductivity, while CoHex and daunomycin do not. Latest results regarding the reproducible frequency responses indicate considerable isotope effects also in EQCM measurements depending on the redox molecule interacting with the ssDNA‐SAM. These effects will provide new opportunities in drug screening and studies of DNA damage by toxic chemicals.

This communication reports on electrochemical detection of thrombin based on labeling with osmium tetroxide bipyridine [OsO4(bipy)]. Tryptophan amino acids can be labeled at the C−C‐double bond, and at least some tryptophan moieties are accessible for labeling in thrombin. Using the catalytic hydrogen signal from adsorptive stripping voltammetry performed on hanging mercury drop electrode, we could detect as little as 1.47 nM [OsO4(bipy)]‐modified thrombin. We also tested the binding of [OsO4(bipy)]‐modified thrombin with the classic thrombin binding aptamer (TBA) on gold electrodes. This preliminary study revealed that even after modification, a major part of the affinity was conserved, and that the aptamer self‐assembled monolayer (SAM) could be regenerated several times. Molecular simulations confirm that [OsO4(bipy)]‐modified thrombin largely preserves the high binding affinity also of the alternative HD22 aptamer to thrombin, albeit at slightly reduced affinities due to steric hindrance when tryptophans 96 and 237 are labelled. Based on these simulations, compensatory modifications in the aptamer should result in significantly improved binding with labelled thrombin. This combined experimental‐computational approach lays the groundwork for the rational design of improved aptamer sensors for analytical applications.

Biomolecular condensates form via multivalent interactions among key macromolecules and are regulated through ligand binding and/or post-translational modifications. One such modification is ubiquitination, the covalent addition of ubiquitin (Ub) or polyubiquitin chains to target macromolecules for various cellular processes. Specific interactions between polyubiquitin chains of different linkages and partner proteins, including hHR23B, NEMO, and UBQLN2, regulate condensate assembly or disassembly. Here, we used a library of designed polyubiquitin hubs and UBQLN2 as model systems for determining the driving forces of ligand-mediated phase transitions. Systematic decreases to the binding affinity between Ub and UBQLN2 or deviations from the optimal spacing between Ub units reduce the ability of hubs to modulate UBQLN2 phase behavior. Using an analytical model that accurately described the effects of different hubs on UBQLN2 phase diagrams, we determined that introduction of Ub to UBQLN2 condensates incurs a significant inclusion energetic penalty. This penalty competes with the hub's ability to scaffold multiple UBQLN2 molecules, thereby cooperatively amplifying phase separation. Importantly, there exists an optimal polyubiquitin hub design that maximally promotes phase separation. Hubs where Ub units are too close or too far apart inhibit phase separation. These effects on phase diagrams are encoded in the spacings between Ub units as found for naturally-occurring chains of different linkages and designed chains of different architectures, thus illustrating how the ubiquitin code regulates functionality via the emergent properties of the condensate. We expect our findings to extend to other condensates necessitating the consideration of ligand properties, including concentration, valency, affinity, and spacing between binding sites in studies and designs of condensates.

This paper describes the largest H/D kinetic isotope effect (KIE) at room temperature (25 °C) reported to date (kH/kD=2400). In voltammetric measurements with DNA monolayers on gold electrodes, a maximum shift of −400 mV was recorded for the reduction peak potential of 50 μM hexammine cobalt(III) (CoHex) in 10 mM Tris buffer upon replacing water with deuterium oxide in the electrolyte. In subsequent comparative investigations, a much smaller shift (ca. −80 mV) was recorded with daunomycin, whereas no potential shift was recorded with hexammine ruthenium(III) (RuHex). The interactions of RuHex, CoHex, and daunomycin with a mixed self‐assembled monolayer of single‐stranded DNA and 6‐mercapto‐1‐hexanol (ssDNA/MCH SAM) immobilized on gold electrodes were studied by using cyclic and differential‐pulse voltammetry (CV and DPV, respectively). The hydrogen‐bond network within the ssDNA layer seems to amplify the voltammetric H/D isotope effect with CoHex. Voltammetric studies of H/D isotope effects can provide a platform to investigate amplified isotope effects probably not only on DNA layers, but also on proteins and small organic molecules and may be useful for studies of cell proliferation as well as drug testing.

We compare the thermoelectrochemical behavior of paracetamol (acetaminophen) at both directly heated gold micro‐wire electrodes (1–115 °C) and rotating disk electrodes (100–8000 rpm). Koutecky‐Levich plots were recorded at various temperatures between 23 and 60 °C. The calculated Ik values (representing reaction rate at infinite mass transport) were plotted as Arrhenius‐type graphs, and the obtained energy values of 20–30 kJ/mol depended on the potential chosen for the corresponding Koutecky‐Levich plots. This led to the conclusion that these energy values are not activation energies, but rather an energy difference due to overvoltage as described in the Butler‐Volmer equation. Accordingly, Koutecky‐Levich plots recorded at various temperatures allowed to determine the Butler‐Volmer kinetics including transfer coefficients even at low concentrations. Arrhenius plots obtained at heated micro‐wire electrodes revealed two linear regions corresponding to diffusion control with 16 kJ/mol at high temperature and 20–30 kJ/mol at very low temperature. The latter activation energy values were found in a very narrow temperature range and probably belong to a transition region towards a kinetic activation energy.

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