Protein condensates as aging Maxwell fluids

bioRxiv (Cold Spring Harbor Laboratory), 2022

Biomolecular condensates are important contributors to the internal organization of the cell material. While initially described as liquid-like droplets, the term biomolecular condensates is now used to describe a diversity of condensed phase assemblies with material properties extending from low to high viscous liquids, gels, and even glasses. Because the material properties of condensates are determined by the intrinsic behaviour of their molecules, characterising such properties is integral to rationalising the molecular mechanisms that dictate their functions and roles in health and disease. Here, we apply and compare three distinct computational methods to measure the viscoelasticity of biomolecular condensates in molecular simulations. These methods are the shear stress relaxation modulus integration (SSRMI), the oscillatory shear (OS) technique, and the bead tracking (BT) method. We find that, although all of these methods provide consistent results for the viscosity of the condensates, the SSRMI and OS techniques outperform the BT method in terms of computational efficiency and statistical uncertainty. We, thus, apply the SSRMI and OS techniques for a set of 12 different protein/RNA systems using a sequence-dependent high-resolution coarse-grained model. Our results reveal a strong correlation between condensate viscosity and density, as well as with protein/RNA length and the number of stickers vs. spacers in the amino-acid protein sequence. Moreover, we couple the SSRMI and the OS technique to nonequilibrium molecular dynamics simulations that mimic the progressive liquid-to-gel transition of protein condensates due to the accumulation of inter-protein β-sheets. We compare the behaviour of three different protein condensates-i.e., those formed by either hnRNPA1, FUS, or TDP-43 proteins-whose liquid-to-gel transitions are associated with the onset of amyotrophic lateral sclerosis and frontotemporal dementia. We find that both SSRMI and OS techniques successfully predict the transition from functional liquid-like behaviour to kinetically arrested states once the network of inter-protein β-sheets has percolated through the condensates. Overall, our work provides a comparison of different modelling rheological techniques to assess the viscosity of biomolecular condensates, a critical magnitude that provides information on the behaviour of biomolecules inside condensates.

2024

Biomolecular condensates are dynamic intracellular entities defined by their sequence-and compositionencoded material properties. During aging, these properties can change dramatically, potentially leading to pathological solidlike states, the mechanisms of which remain poorly understood. Recent experiments reveal that the aging of condensates involves a complex interplay of solvent depletion, strengthening of sticker links, and the formation of rigid structural segments such as beta fibrils. In this study, we use various coarse-grained models to investigate how solvent expulsion, biopolymer chain rigidity, and the lifetimes of sticker contacts influence the viscoelastic properties and aging dynamics of condensates. We find that the rigidity of the biopolymer backbone is essential for replicating the predominant elastic behavior observed in experiments. In contrast, models using fully flexible chains-an assumption common in simulations of intrinsically disordered proteins-fail to exhibit a dominant elastic regime. We also demonstrate that altering the solvent content within condensates affects the crossover between storage and loss moduli. This suggests that desolvation plays a significant role in condensate aging by promoting the transition from a viscous to an elastic state. Furthermore, the lifetime of sticker pairs profoundly influences the mature state of the condensates; short-lived stickers lead to a Maxwell fluid behavior, while longer-lived, irreversibly cross-linked stickers result in solidlike properties, consistent with the Kelvin-Voigt model. Finally, by incorporating the chain rigidification, desolvation, and sticker pair formation into a nonequilibrium dynamic aging simulation, we show the molecular mechanism of forming solid shells around the condensate surfaces observed in a recent experimental report.

Physical Review Letters, 2010

The magnetically driven rotation of 300 nm diameter rods shows the surface viscosity of albumin at an air-water interface increases from 10 À9 to 10 À5 N s=m over 2 h while the surface pressure saturates in minutes. The increase in surface viscosity is not accompanied by a corresponding increase in elasticity, suggesting that the protein film anneals with time, resulting in a more densely packed film leading to increased resistance to shear. The nanometer dimensions of the rods provide the same sensitivity as passive microrheology with an improved ability to measure more viscous films.

Food Hydrocolloids, 1999

The effect of ageing of the protein monolayer around the droplets on the rheology of a heat-set whey protein emulsion gel has been investigated at neutral pH. A fine β-lactoglobulin-stabilized emulsion (30vol.% oil) was either mixed immediately with whey protein isolate or left for up to few days before mixing and subsequent heat treatment (85°C for 35min). It was found

Soft Matter, 2011

Globular proteins influence the flow, microstructure, phase behavior and transport of biofluids and biomolecules in the mammalian body. These proteins are essential constituents of food, drugs and cosmetics, and their dynamics determine the physical properties and application of these multicomponent materials. In conventional rheological studies conducted using typical geometries on torsional rheometers, solutions of globular proteins are commonly reported to have a solid-like response at concentrations as low as 0.03% by weight. Typical explanations invoke the presence of long-range repulsions that are stronger than electrostatic interactions. In this study, we probe the bulk and the interfacial viscoelasticity of surfactant-free bovine serum albumin (BSA) solutions using a stress-controlled torsional rheometer, augmented by microfluidic rheometry and interfacial rheometric measurements. We demonstrate that the origin of this yield-like behavior, which is manifested as a highly shear-thinning bulk rheological response, lies in the formation of a film of adsorbed protein, formed spontaneously at the solution/gas interface. We provide direct interfacial rheometric measurements to study the concentration-dependent viscoelasticity of the adsorbed protein and we describe a simple, but quantitative, additive model useful for extracting the interfacial viscosity contribution from bulk viscosity measurements over a wide range of shear rates.

Physical Review Letters, 2018

An increasing number of proteins with intrinsically disordered domains have been shown to phase separate in buffer to form liquid-like phases. These protein condensates serve as simple models for the investigation of the more complex membrane-less organelles in cells. To understand the function of such proteins in cells, the material properties of the condensates they form are important. However, these material properties are not well understood. Here, we develop a novel method based on optical traps to study the frequency-dependent rheology and the surface tension of PGL-3 condensates as a function of salt concentration. We find that PGL-3 droplets are predominantly viscous but also exhibit elastic properties. As the salt concentration is reduced, their elastic modulus, viscosity and surface tension increase. Our findings show that salt concentration has a strong influence on the rheology and dynamics of protein condensates suggesting an important role of electrostatic interactions for their material properties. Here, the tilde denotes the complex amplitude of the Fourier mode at angular frequency ω and c.c. refers to the complex conjugate. In Eq. (2), χ * (ω) = χ (ω) + iχ (ω)

2020

A wide range of systems containing proteins have been shown to undergo liquid-liquid phase separation (LLPS) forming membraneless compartments, such as processing bodies1, germ granules2, stress granules3 and Cajal bodies4. The condensates resulting from this phase transition control essential cell functions, including mRNA regulation, cytoplasm structuring, cell signalling and embryogenesis1–4. RNA-binding Fused in Sarcoma (FUS) protein is one of the most studied systems in this context, due to its important role in neurodegenerative diseases5–7. It has recently been discovered that FUS condensates can undergo an irreversible phase transition which results in fibrous aggregate formation6. Gelation of protein condensates is generally associated with pathology. One case where liquid-to-solid transition (LST) of liquid-liquid phase separated proteins is functional, however, is that of silk spinning8,9, which is largely driven by shear, but it is not known what factors control the path...

Journal of Colloid and Interface Science, 1997

3) . Configurational changes and physical instability of The purpose of this study was to investigate and compare inbulk protein molecules may result as consequences of enviterfacial rheological and tension properties of adsorbed protein ronmental changes, including pH, ionic strength, temperafilms under conditions known to affect bulk properties of proteins. ture, aging, protein concentration, and the addition of certain In previous publications, the effects of pH, protein concentration, chemical agents (3 -5) . Urea, guanidine hydrochloride, and temperature, and aging on the interfacial rheology of bovine serum copper sulfate are examples of protein chemical denaturants albumin (BSA) and human immunoglobulin G (HI g G) were re-( 5) . These molecules bind to proteins, resulting in loss of ported (tertiary and quaternary structure. pH affects the ionization Sci. Technol. 45, 239 (1991); and D. J. Burgess, J. K. Yoon, and N. O. Sahin, J. Parenteral Sci. Technol. 46, 150 ). These of the carboxyl and amino groups of proteins, which in turn data are compared with interfacial tension data reported here. In may affect intramolecular interaction and hence folding (3, addition, the effects of ionic strength and chemical agents on the 4 ). The effective charge on proteins is dependent on the interfacial rheology and tension of BSA and HI g G are reported. ionic strength through counterion screening, and conse-An oscillatory interfacial shear rheometer was used to determine quently ionic strength alters molecular folding. Change in interfacial rheology, and a Cahn microbalance connected to a Wilthe molecular thermal energy of proteins can result in conhelmy plate was used to measure interfacial tension. These two formational change (3 ), which can be reversible or irreverstechniques provide information on molecular interfacial adsorpible, depending on the temperature. High temperatures can tion, interaction between adsorbed molecules, film compactness, induce bond cleavage and hence irreversible denaturation.

Nature communications, 2024

Material properties of phase-separated biomolecular condensates, enriched with disordered proteins, dictate many cellular functions. Contrary to the progress made in understanding the sequence-dependent phase separation of proteins, little is known about the sequence determinants of condensate material properties. Using the hydropathy scale and Martini models, we computationally decipher these relationships for charge-rich disordered protein condensates. Our computations yield dynamical, rheological, and interfacial properties of condensates that are quantitatively comparable with experimentally characterized condensates. Interestingly, we find that the material properties of model and natural proteins respond similarly to charge segregation, despite different sequence compositions. Molecular interactions within the condensates closely resemble those within the single-chain ensembles. Consequently, the material properties strongly correlate with molecular contact dynamics and single-chain structural properties. We demonstrate the potential to harness the sequence characteristics of disordered proteins for predicting and engineering the material properties of functional condensates, with insights from the dilute phase properties. Material properties of biomolecular condensates play a key role in the proper execution of different biological functions, e.g., cell division 1 , selective autophagy 2,3 , gene regulation 4,5 , and nuclear-cytoplasmic shuttling . In general, the material state of condensates ranges from reversible liquid-like assemblies with functional relevance to irreversible solid-like assemblies with pathological consequences . The viscous nature (liquidity) of the condensates formed via phase separation enables them to act as dynamic assemblies, which exchange molecules with the surrounding environment, and dissolve as required, in response to physiological cues . To better understand the spatiotemporal evolution of condensates, researchers have recently started characterizing their mesoscopic material properties such as diffusion coefficient, viscosity, viscoelasticity, and surface tension through experiments . However, much remains unknown, especially how the protein sequence dictates these different material properties. Intrinsically disordered proteins (IDPs) or regions (IDRs) are deemed essential for condensate formation , which across proteomes, typically consist of a high fraction of charged residues . Alterations within an IDP sequence, e.g., due to mutation or posttranslational modification, can lead to changes in the intra-and intermolecular interactions, which can impact both the phase behavior and material properties of the condensates formed or can provide the ability to modulate them independently 24 . For example, changes in the

Journal of Chemical Physics, 2003

The mechanism of protein stabilization by glassy solvents is not entirely clear, and the stabilizer effective for a given protein is often discovered empirically. We use low frequency Raman spectroscopy as an effective tool to directly evaluate the ability of different solvents to suppress the conformational fluctuations that can lead to both protein activity and denaturation. We demonstrate that while trehalose provides superior suppression at high temperatures, glycerol is more effective at suppressing protein dynamics at low temperatures. These results suggest that viscosity of the solvent is not the only parameter important for biopreservation. It is also shown that glycerol and water enhance the high temperature conformational fluctuations relative to dry lysozyme, which explains the lower melting temperatures T m in the hydrated protein and protein formulated in glycerol.