Electrochemical gradients are essential to the functioning of cells and form across membranes using active transporters. Here we show in contrast that condensed biomolecular systems—often termed condensates—sustain pH gradients without any external energy input. By studying individual condensates on the micrometre scale using a microdroplet platform, we reveal dense-phase pH shifts towards conditions of minimal electrostatic repulsion. We demonstrate that protein condensates can drive substantial alkaline and acidic gradients, which are compositionally tunable and can extend to complex architectures sustaining multiple unique pH conditions simultaneously. Through in silico characterization of human proteomic condensate networks, we further highlight potential wide-ranging electrochemical properties emerging from condensation in nature, while correlating intracellular condensate pH gradients with complex biomolecular composition. Together, the emergent nature of condensation shapes distinct pH microenvironments, thereby creating a regulatory mechanism to modulate biochemical activity in living and artificial systems.

The calcium-binding protein S100A9 is recognized as an important component of the brain neuroinflammatory response to the onset and development of neurodegenerative disease. S100A9 is intrinsically amyloidogenic and in vivo co-aggregates with amyloid-β peptide and α-synuclein in Alzheimer’s and Parkinson’s diseases, respectively. It is widely accepted that calcium dyshomeostasis plays an important role in the onset and development of these diseases, and studies have shown that elevated levels of calcium limit the potential for S100A9 to adopt a fibrillar structure. The exact mechanism by which calcium exerts its influence on the aggregation process remains unclear. Here we demonstrate that despite S100A9 exhibiting α-helical secondary structure in the absence of calcium, the protein exhibits significant plasticity with interconversion between different conformational states occurring on the micro- to milli-second timescale. This plasticity allows the population of conformational states that favour the onset of fibril formation. Magic-angle spinning solid-state NMR studies of the resulting S100A9 fibrils reveal that the S100A9 adopts a single structurally well-defined rigid fibrillar core surrounded by a shell of approximately 15–20 mobile residues, a structure that persists even when fibrils are produced in the presence of calcium ions. These studies highlight how the dysregulation of metal ion concentrations can influence the conformational equilibria of this important neuroinflammatory protein to influence the rate and nature of the amyloid deposits formed.

The hexameric AAA+ protein ClpC, combined with peptidase ClpP, forms a critical ATP-dependent protease in bacteria, essential for virulence. ClpC is usually repressed in an inactive resting state, where two ClpC spirals interact via coiled-coil M-domains. Antibacterial peptides and partner proteins trigger ClpC activation by binding to its N-terminal domain (NTD). This study reveals that the NTD stabilizes the resting state through multiple anchoring points to M-domains and ATPase domains. The same NTD sites also serve as binding sites for adaptor proteins and substrates carrying phosphorylated arginines (pArg), disrupting resting state interactions and promoting active ClpC hexamer formation. This coupling ensures that ClpC activation aligns with substrate and partner protein availability. Toxic peptides exploit this regulatory mechanism, leading to continuous ClpC activation and harmful, uncontrolled proteolysis. These findings highlight the dual role of the NTD in maintaining resting state stability and mediating activation, emphasizing its critical role in bacterial protease regulation and its potential as a drug target.

Neurofibrillary tangles (NFTs) formed from the protein tau disrupt neuronal function in Alzheimer’s disease and are strongly associated with cognitive decline. Early events in tau aggregation are increasingly linked to the formation of biomolecular condensates, which lower the energetic barriers to pathological aggregation by acting as intermediates that transition into insoluble assemblies, a mechanism also implicated in other neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Despite growing evidence for this pathway, the molecular basis by which reversible condensates evolve into irreversible, pathogenic aggregates has remained unclear. Here, we map the phase behavior, structural transitions, and thermodynamic reversibility of tau during condensate aging. Our results reveal that the two hallmark features of the pathological end state, β-sheet enrichment and irreversible aggregation, emerge at different rates and occupy distinct regions of the phase space, indicating that these properties are mechanistically uncoupled. Notably, we identify tau condensate phases that are β-sheet rich yet thermodynamically reversible, as well as irreversible intermediates that lack β-sheet structure. These findings expand the landscape of tau aggregate species beyond a simple linear progression toward fibrils and highlight a diverse array of intermediates with distinct structural and thermodynamic properties. This decoupling of structure and irreversibility has important implications for understanding tau aggregation mechanisms and may offer targets for therapeutic intervention.

Neuronal cell death induced by cell membrane damage is one of the major hallmarks of neurodegenerative diseases. Neuroinflammation precedes the loss of neurons; however, whether and how inflammation-related proteins contribute to the loss of membrane integrity remains unknown. We employed a range of biophysical tools, including high-speed atomic force microscopy, fluorescence spectroscopy, and electrochemical impedance spectroscopy, to ascertain whether the pro-inflammatory protein S100A8 induces alterations in biomimetic lipid membranes upon interaction. Our findings underscore the crucial roles played by divalent cations and membrane charge. We found that apo-S100A8 selectively interacts with anionic lipid membranes composed of phosphatidylserine (PS), causing membrane disruption through a detergent-like mechanism, primarily affecting regions where phospholipids are less tightly packed. Interestingly, the introduction of Ca²⁺ ions inhibited S100A8-induced membrane disruption, suggesting that the disruptive effects of S100A8 are most pronounced under conditions mimicking intracellular compartments, where calcium levels are low, and PS concentrations in the inner leaflet of the membrane are high. Overall, our results present a mechanistic basis for understanding the molecular interactions between S100A8 and the plasma membrane, emphasizing S100A8 as a potential contributor to the onset of neurodegenerative diseases.

The S100 family consists of calcium binding proteins that are largely known for their contribution to the neuroinflammatory processes. They are associated with various cardiac and neurological functions as well as related diseases. A few S100 proteins can form unspecific or amyloid aggregates in neuropathologies and thus play a part in dementia pathogenesis. Among all S100 proteins, S100B and S100A9 aggregation properties are the most investigated, however, there is a lack of studies regarding other S100 members. In particular, S100A1 and S100A8 are also associated with neuropathies, but their interactions or aggregation are poorly understood. Therefore, in this study, we explored whether S100A1 and S100A8 proteins can form heterodimers, interact or co-aggregate. Our results revealed that S100A1 and S100A8 interactions and amyloid aggregation are driven by calcium ions. We observed that while S100A1 remains mostly stable, S100A8 forms various types of spherical or unspecific aggregates. While they do not form stable heterodimers like calprotectin, their transient interactions facilitate the formation of worm-like amyloid fibrils and the process is regulated by different calcium ion concentrations. At calcium ions saturation, both proteins are stabilized leading to inhibition of aggregation. Overall, by employing a diverse range of techniques from amyloid and protein-specific fluorescence detection to electron-electron double resonance spectroscopy, we elucidated interactions between S100 proteins that might otherwise be overlooked, enhancing our understanding of their aggregation behaviour.