Biomolecular Condensates Dictate the Folding Landscape of Protein Alpha-Helices

Chain Collapse, Reduced Dielectric, and Water Release Drive Protein Phase Separation.

Biomolecular condensates represent unique microenvironments that organize intracellular biology and promote biochemical reactions. However, the biomolecular interactions driving condensate phase separation are often weak, transient, and heterogeneous. Investigating the structural biology and chemical properties of condensate interiors has therefore proven experimentally challenging, often requiring the use of perturbative probes. To overcome this challenge, we combine label-free optical scattering and vibrational spectroscopy approaches spanning ultraviolet, visible, mid-infrared, and terahertz wavelengths with deep-learning-based ensemble prediction of intrinsically disordered protein conformations. This suite of label-free approaches provides quantitative insights into protein-protein/protein-solvent interactions and the chemical properties of condensate interiors. Investigating the N-terminal domain of the RNA DEAD-box helicase 4 (DDX4), our experimental and computational results support a model of phase separation involving protein chain collapse, reduced dielectric, and water release. These molecular events are expected to enhance the strength of multivalent protein-protein interactions within condensates, creating a positive feedback loop important for condensate growth and phase separation.

Dual roles of condensates in protein misfolding and aggregation.

Protein aggregation is a hallmark in several neurodegenerative diseases, in which proteins assemble into structurally diverse misfolded states, ranging from amorphous aggregates to amyloid fibrils. Several aggregation-prone proteins can undergo phase separation to form biomolecular condensates, creating distinct chemical environments compared to the surrounding dilute phase. These environments dictate protein conformations, interaction networks, and free energy landscapes, thereby modulating aggregation pathways. Notably, condensates exert dual and context-dependent effects: they can promote aggregation by stabilizing misfolded intermediates and facilitating assembly, or they can suppress aggregation by buffering interactions and retaining proteins. Here, we summarize the current literature and describe biomolecular condensates as key regulators of protein misfolding and aggregation and highlight the importance of the local milieu in determining aggregation outcomes.

Structure, Diffusion, and Relaxation in a Charge-Neutral ProTalpha-Histone H1 Condensate

Condensates formed by oppositely charged intrinsically disordered proteins provide model systems for understanding how transient electrostatic interactions govern structure and dynamics in biomolecular assemblies. Here we investigate a nearly charge-neutral condensate composed of 50 Prothymosin alpha (ProTalpha) and 40 Histone H1 molecules using a single-bead-per-residue coarse-grained model combining the HPS hydropathy model for disordered regions with a Go model for the globular domain of Histone H1 under NPT conditions at pressures from 2 to 12 bar. We find that chain dimensions, including the radius of gyration (Rg), end-to-end distance (Ree), and their ratio R, are insensitive to pressure, indicating that chain conformations remain largely unchanged over the pressure range studied. Histone H1 exhibits systematically larger values of R than ProTalpha because of its globular-core plus disordered-tail architecture. Translational diffusion coefficients decrease monotonically with pressure, from approximately 0.22 to 0.06 nm^2/ns, with substantial chain-to-chain heterogeneity comparable to the mean diffusion coefficient. Chain relaxation follows a stretched exponential with beta less than 1 that decreases with pressure. ProTalpha relaxation times of approximately 12 to 40 ns obey Rouse scaling, whereas Histone H1 deviates because of the internal constraint imposed by its globular domain. ProTalpha-Histone H1 contact lifetimes of approximately 0.43 to 0.56 ns are much shorter than the Rouse relaxation time, placing the system firmly in the fast-exchange regime where transient electrostatic contacts renormalize chain friction rather than acting as permanent cross-links, consistent with the moderate stretching exponent beta of approximately 0.55 to 0.70 observed across all pressures.

Proteins in action : simulations of conformational changes in small proteins

Programming Multidomain Peptides With Molecular Frustration Into Biomolecular Condensates

The discovery of biomolecular condensates, driven by liquid–liquid phase separation of intrinsically disordered proteins has significant impacts on both fundamental and applied science and engineering. Although most studies on biomolecular condensates focus on intrinsically disordered structures, research on the role of molecular ordering remains largely unexplored, however is beneficial for gaining new mechanistic understanding and further expand the design space of peptides for constructing functional condensates. Toward this goal, we conducted systematic studies on how molecular ordering impacts the phase behaviors of peptides using multidomain peptides (MDPs) as a model system. MDPs were designed using a molecular frustration principle in which parts of the peptides favored β-sheet assembly and parts favored disassembly. Through programming of each domain, it is evident that the phase behavior of MDPs is largely dictated by the secondary structure, and partially folded β-sheet plays a key role in driving MDPs to form condensates. We also discovered complex coacervates formed by MDPs and synthetic anionic polymers, which exhibited dramatically improved stability. Furthermore, we show enzyme-triggered condensation can be achieved using phosphorylated MDPs as the molecular precursor and alkaline phosphatase as a molecular switch, highlighting the potential of these materials for bacterial imaging and antimicrobial therapy development.

Local cooperative interactions reshape the folding transition in a one-dimensional spin-glass model

Protein folding is the process by which a polypeptide chain organizes into its three-dimensional structure through a balance of stabilizing and destabilizing interactions encoded by the sequence. A central question in protein biophysics is how thermodynamic factors guide a polypeptide toward its native folded state despite the rugged energy landscape and the competing influence of nonnative interactions. In many biomolecular processes, cooperativity provides a mechanism by which multiple weak interactions act collectively to generate a robust response. In the context of protein folding, such cooperative effects may arise when the formation of one native contact enhances the stability or likelihood of nearby native contacts, thereby promoting collective organization toward the folded state. At the same time, folding is opposed by the much larger number of non-native interactions, whose heterogeneity can introduce frustration and destabilize folding even when the average native bias favors the folded phase. The interplay of these competing effects in determining foldability remains unclear in statistical-mechanical models. Here, we address this problem using a one-dimensional spin-glass model of protein folding with explicit shared-residue cooperative interactions encoded through wedge-based motifs. We show that modest cooperative bias can stabilize folding even where the noncooperative system remains unfolded, whereas non-native energetic fluctuation suppresses folding and shifts the transition to higher cooperative strengths. We further find that partial cooperative coverage is sufficient to lower the folding threshold. Therefore, the model provides a mean-field framework for incorporating cooperative interaction strength into the native one-dimensional model of protein folding and for describing how local cooperativity reshapes the folding transition.