Jul 2026· Current Opinion in Structural Biology· Vol 99, pp.
103324
· 0 citations· 70 references
Medicine
TL;DR
This review emphasizes the context-dependent role of crowding in balancing native protein stability against aggregation propensity, highlighting how heterogeneous intracellular environments govern the emergence of functional assemblies or pathological amyloids.
Abstract
Macromolecular crowding defines the cellular interior, where high biomolecule concentrations reshape protein folding landscapes and intermolecular interactions. Excluded-volume effects, quinary interactions, and spatial confinement collectively modulate amyloidogenic self-assembly, a process central to both functional organization and protein misfolding diseases. This review emphasizes the context-dependent role of crowding in balancing native protein stability against aggregation propensity, highlighting how heterogeneous intracellular environments govern the emergence of functional assemblies or pathological amyloids. Recent computational advances provide key mechanistic insights across scales. Atomistic simulations resolve detailed conformations and interactions, while coarse-grained and implicit condensate models enable exploration of larger systems, longer timescales, phase behavior, and collective assembly. Together, these multiscale approaches underscore the need to move beyond dilute approximations. Integrative frameworks that incorporate enhanced sampling and artificial intelligence are essential to capture cellular complexity and enable predictive understanding of protein self-assembly and amyloidogenesis under crowded conditions.
Macromolecular crowding can reshape the conformational landscapes of intrinsically disordered and marginally stable proteins linked to neurodegeneration, enriching aggregation-prone states and changing how nucleation begins. Crowding can also promote liquid-liquid phase separation, leading to condensates that concentrate proteins, reshape interaction networks, and in some cases promote liquid-to-solid transitions into amyloid assemblies. This crowding-liquid-liquid phase separation-aggregation continuum may help explain why dilute solution assays often fail to capture the mechanisms that operate in cells. Crowding-aware structural biology, including in-cell nuclear magnetic resonance, cryo-electron microscopy of condensates, single-molecule methods, and thermodynamic-kinetic modelling, will be important for resolving physiologically relevant intermediates. From a therapeutic perspective, targeting condensate properties and early oligomeric states, rather than focussing only on mature fibrils, may offer new ways to limit pathogenic aggregation.
Tejas Nikam, Shashi Prakash Patel, S. Saraf et al.· Current Opinion in Structura...· 1 citation
This work investigates how protein folding landscapes are altered inside condensates, using the protein α-helix as a model folded domain and develops a chemically specific, residue-resolution model for quantification of α-helical folding and applies it to characterize diverse helices within condensates of varying physicochemical properties.
Nathaniel Hess, Jerelle A. Joseph· Journal of the American Chem...· 0 citations
Molecular crowding in cells is not solely a consequence of excluded volume but emerges from interaction networks shaped by RNA and proteins. Here we examine the physicochemical principles underlying RNA-mediated crowding, focusing on how sequence-driven multivalency, structural topology, and network connectivity govern molecular organization. Repeats, secondary structures, and higher-order motifs such as G-quadruplexes act as interaction modules that promote percolation and phase separation, while RNA length and concentration tune phase boundaries. These RNA-encoded features promote multivalent RNA-RNA and RNA-protein interactions that shape condensate assembly, dynamics, and organization across scales. In this framework, crowding emerges as an RNA-centered, interaction-driven property linking molecular features to mesoscale organization in both physiological and pathological contexts.
Jonathan Fiorentino, Michele Monti, Laura Broglia et al.· Current Opinion in Structura...· 0 citations
The current literature is summarized and biomolecular condensates are described as key regulators of protein misfolding and aggregation and the importance of the local milieu in determining aggregation outcomes is highlighted.
Emre Pekbilir, Dorothee Dormann· Current Opinion in Structura...· 0 citations
The crowded intracellular milieu shapes the thermodynamics and kinetics of biochemical reactions. RNA, an abundant and structurally versatile polymer, contributes to this crowding by acting both as a physical agent that restricts diffusion and enhances excluded volume, and as a sequence- and structure-specific scaffold driving multivalent RNA-RNA and RNA-protein interactions. These properties position RNA as a biologically active determinant of intracellular organization, distinct from inert synthetic crowders and purely structural scaffolds. This minireview examines how RNA shapes the formation, composition, and material properties of biomolecular condensates, highlighting the molecular grammar encoded in RNA sequence, length and valency, structure, and chemical modifications. We discuss how concentration-dependent, biphasic effects of RNA on condensate assembly can tip the balance between functional compartmentalization and pathological liquid-to-solid transitions implicated in neurodegenerative disease and cancer. Finally, we outline challenges in defining RNA-specific thresholds and translating structural insights into therapeutic strategies for mitigating aberrant RNA-mediated crowding.
Shibam Dey, O. Amster-Choder· Current Opinion in Structura...· 0 citations
Biomolecular condensation has emerged as a central mechanism of cellular organization, regulating fundamental processes from transcription to stress responses. Its dysregulation - often involving transitions from dynamic condensates to more solid or aggregated states - has been linked to human disease and thus represents a growing therapeutic opportunity. Yet, despite substantial progress, precisely relating protein sequence to condensate behavior, function, and dysregulation remains a largely unresolved challenge. Recent advances in high-throughput approaches are beginning to address this gap. By combining large-scale mutagenesis to fitness, fluorescence- or imaging-based selections, and deep sequencing, these methods enable systematic interrogation of different types of protein self-assembly across vast sequence spaces. However, most of the currently available assays measure indirect readouts such as solubility, stability, or cellular fitness and differ in the way they capture different parameters of the self-assembly process. Here, we review emerging high-throughput strategies to study protein condensation and aggregation at scale, emphasizing what they truly measure, their limitations, and how the cross-talk among these complementary approaches can provide a more accurate and mechanistic mapping of sequence-to-assembly relationships.
Mariano Martín, Alice Lissmatz, Benedetta Bolognesi· Current Opinion in Structura...· 0 citations