Matthias Ballauff

Developing insight into liquid–liquid phase separation in complex polyelectrolyte mixtures has acquired new urgency considering the role this process plays in the formation of biomolecular condensates, which are involved in diverse processes including, stress response, signal transduction, metabolism, gene regulation, and many human diseases. Condensates can be viewed as a biological membraneless structure situated, in scale and function, between macromolecules and cells. The physical properties of, and the physico-chemical environments within, condensates are very important determinants of their biological function. These properties and environments are determined by the phase separation process. Earlier experimental work in one of our laboratories characterized in detail a phase separation process in solutions of oppositely charged polypeptides. Considering new information being developed on macromolecular association in homogeneous solutions of oppositely charged polyions, we have revisited our earlier data looking into the influence of intermolecular association on the outcome of the phase transition. Such influence is clearly established in this Brief Report. We demonstrate that dispersity of species in homogeneous solution owing to macromolecular association affects phase separation by creating an open miscibility gap, leading to a dependence of the phase compositions on the initial polymer volume fraction. It is very likely that this plays a significant role in the formation of condensates in the biological context that may have been overlooked.

Glycosaminoglycans (GAGs) are linear, negatively charged biopolymers that modulate complex biological processes, such as blood coagulation, immune regulation, or viral entry. Their sulfation pattern and chain length govern how strongly they bind to other physiologically relevant species. Most of these interactions rely on electrostatic forces facilitated by the strong polyanionic properties of GAGs; therefore, considering these from a polyelectrolyte vantage point can help understand how such charge‐based, often transient interactions contribute to physiological and pathological processes. While the different GAG classes share key electrostatic properties, they exhibit unique structural features that shape their function. Here, it is highlighted on how modern separation and analytical tools exploit the polyanionic character of GAGs to dissect subtle structural details. For these, the fundamental description of their charge–charge interactions is crucial. With this knowledge, modified GAGs, synthetic GAG mimetics, or GAG‐binding molecules can be designed that replicate or refine their key properties and show promise for therapeutic and biomedical applications. Altogether, recognizing the importance of GAGs as polyelectrolytes provides vital knowledge on how their charge distribution mediates crucial biomolecular interactions in health and disease, and thus it helps complete our knowledge on fundamentally important biopolymers.

Glycosaminoglycans (GAGs) are linear, highly acidic polysaccharides that serve as essential extracellular matrix components. There has been increasing evidence that GAGs can release gadolinium ions from complexes of magnetic resonance imaging contrast agents. This unintended release of gadolinium might be an initial step leading to gadolinium deposition disease, as observed in some patients after intravenous injection of such contrast agents. However, the molecular details of the release remain poorly understood. In this work, we provide direct evidence for gadolinium binding by GAGs using synthetic model substance Fondaparinux (FPX), a heparin mimetic. We observed FPX-gadolinium complexes in mass spectrometry experiments and electron paramagnetic resonance spectroscopy (EPR) and characterized the binding by EPR, isothermal titration calorimetry, and gas-phase infrared (IR) spectroscopy. Finally, we were able to follow the transchelation process on a molecular level by utilizing collision-induced dissociation experiments.

Glycosaminoglycans (GAGs) are linear, negatively charged biopolymers that modulate complex biological processes, such as blood coagulation, immune regulation, or viral entry. Their sulfation pattern and chain length govern how strongly they bind to other physiologically relevant species. Most of these interactions rely on electrostatic forces facilitated by the strong polyanionic properties of GAGs; therefore, considering these from a polyelectrolyte vantage point can help understand how such charge-based, often transient interactions contribute to physiological and pathological processes. While the different GAG classes share key electrostatic properties, they exhibit unique structural features that shape their function. Here, we highlight how modern separation and analytical tools exploit the polyanionic character of GAGs to dissect subtle structural details. For these, the fundamental description of their charge-charge interactions is crucial. With this knowledge, modified GAGs, synthetic GAG mimetics, or GAG-binding molecules can be designed that replicate or refine their key properties and show promise for therapeutic and biomedical applications. Altogether, recognizing the importance of GAGs as polyelectrolytes provides an integrative perspective on how their charge distribution mediates crucial biomolecular interactions in health and disease, and they help complete our knowledge on fundamentally important biopolymers.

The thermal unfolding of lysozyme in aqueous solution has been analyzed by (nano) differential scanning fluorimetry (nanoDSF) and differential scanning calorimetry (DSC). In addition, dynamic light scattering (DLS) acquired in parallel to the DSF measurements, was used to confirm that the change in hydrodynamic radius upon unfolding is rather small ( R _H,f =1.75 nm in the folded state; and R _H,u =1.91 nm in the unfolded state). NanoDSF measurements were evaluated to characterize the folding/unfolding transition within the classical two‐state folding model. The temperature of unfolding ( T _m ) is found to be the most robust quantity. The unfolding enthalpy and the change of specific heat were also obtained and errors in the range of 5–10 % and 30–50 % were determined, respectively. A comparison of thermodynamic parameters from nanoDSF and DSC measurements provides evidence for an increasing unfolding enthalpy with protein concentration. A comparison with data from literature suggests that a weak association in the folded state can lead to the observed change of the unfolding enthalpy. For Δc _p significantly higher values is deduced from the analysis of temperature dependent nanoDSF measurements (10 kJ/(K mol)) as compare to DSC (3–5 kJ/(K mol)).

The interaction of unfractionated heparin (UFH) with universal heparin reversal agent 7 (UHRA-7) is investigated. UHRA-7 is composed of a hyperbranched polyglycerol core onto which an array of methylated tris(2-aminoethylamine) (Me-TREN) charged groups is grafted, which in turn are shielded with a layer of small chain poly(ethylene glycol) methyl ether (mPEG) chains. This system has previously been shown to be biocompatible and to be effective at neutralizing heparin. The binding constant K_b was determined from isothermal titration calorimetry experiments, at temperatures ranging from 278 to 323 K and salt concentrations ranging from 0.06 to 0.20 M NaCl. The data were analyzed in terms of a number of different theoretical models to determine the contribution of counterion release and water release to driving the interaction between UFH and UHRA-7. With the support of NMR and molecular dynamics simulation data, a model of the interaction between UFH and UHRA-7 is proposed. The binding of heparin and universal heparin reversal agent 7 is mainly due to charge-charge interactions between the negatively charged units on UFH with positively charged Me-TREN and mPEG chains.

A thermodynamic analysis of the binary complex formation of the highly positively charged linker histone H1 and the highly negatively charged chaperone prothymosin α (ProTα) is detailed. ProTα and H1 have large opposite net charges (−44 and +53, respectively) and form complexes at physiological salt concentrations with high affinities. The data obtained for the binary complex formation are analyzed by a thermodynamic model that is based on counterion condensation modulated by hydration effects. The analysis demonstrates that the release of the counterions mainly bound to ProTα is the main driving force, and effects related to water release play no role within the limits of error. A strongly negative Δc_p (=−0.87 kJ/(K mol)) is found, which is due to the loss of conformational degrees of freedom.

A thermodynamic analysis of the binary complex formation of the highly positively charged linker histone H1 and the highly negatively charged chaperone, prothymosin α (ProTα) is given. ProTα and H1 have large opposite net charges (−44 and +53, respectively) and form complexes at physiological salt concentrations with high affinity as shown by Chowdhury et al. (Proc Natl Acad Sci U S A 2023, 120 (41), e2304036120). Data obtained by Chowdhury et al. for binary complex formation are analyzed by a thermodynamic model that is based on counterion condensation modulated by hydration effects. The analysis demonstrates that the release of the counterions mainly bound to ProT is the main driving force, effects related to water release play no role within the limits of error. A strongly negative cp (= -0.87 kJ/(K mol)) is found which is due to the loss of conformational degrees of freedom.