Biomolecular Phase Research Articles

Driving force of biomolecular liquid–liquid phase separation probed by nuclear magnetic resonance spectroscopy

The assembly of biomolecular condensates is driven by liquid–liquid phase separation. To understand the structure and functions of these condensates, it is essential to characterize the underlying driving forces, <i>e</i>.<i>g</i>., protein–protein and protein–RNA interactions. As both structured and low-complexity domains are involved in the phase separation process, NMR is probably the only technique that can be used to depict the binding topology and interaction modes for the structured and nonstructured domains simultaneously. Atomic-resolution analysis for the intramolecular and intermolecular interactions between any pair of components sheds light on the mechanism for phase separation and biomolecular condensate assembly and disassembly. Herein, we describe the procedures used for the most extensively employed NMR techniques to characterize key interactions for biomolecular phase separation.

The flexibility-based modulation of DNA nanostar phase separation.

Phase separation of biomolecules plays key roles in physiological compartmentalization as well as pathological aggregation. A deeper understanding of biomolecular phase separation requires dissection of a relation between intermolecular interactions and resulting phase behaviors. DNA nanostars, multivalent DNA assemblies of which sticky ends define attractive interactions, represent an ideal system to probe this fundamental relation governing phase separation processes. Here, we use DNA nanostars to systematically study how structural flexibility exhibited by interacting species impacts their phase behaviors. We design multiple nanostars with a varying degree of flexibility using single-stranded gaps of different lengths in the arm of each nanostar unit. We find that structural flexibility drastically alters the phase diagram of DNA nanostars in such a way that the phase separation of more flexible structures is strongly inhibited. This result is not due to self-inhibition from the loss of valency but rather ascribed to a generic flexibility-driven change in the thermodynamics of the system. Our work provides not only potential regulatory mechanisms cells may exploit to dynamically control intracellular phase separation but also a route to build synthetic systems of which assembly can be controlled in a signal dependent manner.

Biomolecular phase separation through the lens of sodium-23 NMR.

Phase separation is a fundamental physicochemical process underlying the spatial arrangement and coordination of cellular events. Detailed characterization of biomolecular phase separation requires experimental access to the internal environment of dilute and especially condensed phases at high resolution. In this study, we take advantage from the ubiquitous presence of sodium ions in biomolecular samples and present the potentials of 23Na NMR as a proxy to report the internal fluidity of biomolecular condensed phases. After establishing the temperature and viscosity dependence of 23Na NMR relaxation rates and translational diffusion coefficient, we demonstrate that 23Na NMR probes of rotational and translational mobility of sodium ions are capable of capturing the increasing levels of confinement in agarose gels in dependence of agarose concentration. The 23Na NMR approach is then applied to a gel‐forming phenylalanine‐glycine (FG)‐containing peptide, part of the nuclear pore complex involved in controlling the traffic between cytoplasm and cell nucleus. It is shown that the 23Na NMR together with the 17O NMR provide a detailed picture of the sodium ion and water mobility within the interior of the FG peptide hydrogel. As another example, we study phase separation in water‐triethylamine (TEA) mixture and provide evidence for the presence of multiple microscopic environments within the TEA‐rich phase. Our results highlight the potentials of 23Na NMR in combination with 17O NMR in studying biological phase separation, in particular with regards to the molecular properties of biomolecular condensates and their regulation through various physico‐ and biochemical factors.

Tunable multiphase dynamics of arginine and lysine liquid condensates

Liquid phase separation into two or more coexisting phases has emerged as a new paradigm for understanding subcellular organization, prebiotic life, and the origins of disease. The design principles underlying biomolecular phase separation have the potential to drive the development of novel liquid-based organelles and therapeutics, however, an understanding of how individual molecules contribute to emergent material properties, and approaches to directly manipulate phase dynamics are lacking. Here, using microrheology, we demonstrate that droplets of poly-arginine coassembled with mono/polynucleotides have approximately 100 fold greater viscosity than comparable lysine droplets, both of which can be finer tuned by polymer length. We find that these amino acid-level differences can drive the formation of coexisting immiscible phases with tunable formation kinetics and can be further exploited to trigger the controlled release of droplet components. Together, this work provides a novel mechanism for leveraging sequence-level components in order to regulate droplet dynamics and multiphase coexistence.

Biomolecular Phase Separation: From Molecular Driving Forces to Macroscopic Properties.

Biological phase separation is known to be important for cellular organization, which has recently been extended to a new class of biomolecules that form liquid-like droplets coexisting with the surrounding cellular or extracellular environment. These droplets are termed membraneless organelles, as they lack a dividing lipid membrane, and are formed through liquid-liquid phase separation (LLPS). Elucidating the molecular determinants of phase separation is a critical challenge for the field, as we are still at the early stages of understanding how cells may promote and regulate functions that are driven by LLPS. In this review, we discuss the role that disorder, perturbations to molecular interactions resulting from sequence, posttranslational modifications, and various regulatory stimuli play on protein LLPS, with a particular focus on insights that may be obtained from simulation and theory. We finally discuss how these molecular driving forces alter multicomponent phase separation and selectivity.

Finite-size scaling analysis of protein droplet formation.

The formation of biomolecular condensates inside cells often involve intrinsically disordered proteins (IDPs), and several of these IDPs are also capable of forming dropletlike dense assemblies on their own, through liquid-liquid phase separation. When modeling thermodynamic phase changes, it is well known that finite-size scaling analysis can be a valuable tool. However, to our knowledge, this approach has not been applied before to the computationally challenging problem of modeling sequence-dependent biomolecular phase separation. Here we implement finite-size scaling methods to investigate the phase behavior of two 10-bead sequences in a continuous hydrophobic-polar protein model. Combined with reversible explicit-chain Monte Carlo simulations of these sequences, finite-size scaling analysis turns out to be both feasible and rewarding, despite relying on theoretical results for asymptotically large systems. While both sequences form dense clusters at low temperature, this analysis shows that only one of them undergoes liquid-liquid phase separation. Furthermore, the transition temperature at which droplet formation sets in is observed to converge slowly with system size, so that even for our largest systems the transition is shifted by about 8%. Using finite-size scaling analysis, this shift can be estimated and corrected for.

Studying RNA Modulated Protein Liquid-Liquid Phase Separation using Coarse-Grained Models

Disordered proteins phase separate in biological cells creating distinct membraneless organelles coexisting with the surrounding cytosol. However, living cells contain a multitude of proteins, nucleic acids, and lipids with remarkable spatiotemporal organization controlling their interactions for specific functions. Experiments have found that membraneless organelles such as P granules are mixtures of RNA and proteins that form via phase separation. Hence, it is essential to study biomolecular phase separation, taking into account the presence of RNA for a deeper understanding of the in vivo formation and function of membraneless organelles. We have successfully studied protein phase separation via molecular dynamics, using a coarse-grained (CG) modeling approach where we effectively capture protein-protein interactions at the amino acid level. In this work, we extend our CG approach to incorporate RNA and determine how it modulates protein phase separation. We directly simulate the co-phase separation of both the disordered domain of DEAD-box helicase protein LAF-1 and polyA RNA co-localized in a slab geometry using standard molecular dynamics simulations conducted over a range of temperatures. We find that favorable RNA-protein interactions lead to the formation of condensed phases containing both RNA and protein, which is consistent with the experiment. The presence of RNA lowers the concentration of protein in the dense phase, and RNA-protein interactions also change the dynamics of proteins inside the condensed phase.

Biomolecular condensates in neurodegeneration and cancer.

The intracellular environment is partitioned into functionally distinct compartments containing specific sets of molecules and reactions. Biomolecular condensates, also referred to as membrane-less organelles, are diverse and abundant cellular compartments that lack membranous enclosures. Molecules assemble into condensates by phase separation; multivalent weak interactions drive molecules to separate from their surroundings and concentrate in discrete locations. Biomolecular condensates exist in all eukaryotes and in some prokaryotes, and participate in various essential house-keeping, stress-response and cell type-specific processes. An increasing number of recent studies link abnormal condensate formation, composition and material properties to a number of disease states. In this review, we discuss current knowledge and models describing the regulation of condensates and how they become dysregulated in neurodegeneration and cancer. Further research on the regulation of biomolecular phase separation will help us to better understand their role in cell physiology and disease.

Chain stiffness bridges conventional polymer and bio-molecular phases.

Chain molecules play important roles in industry and in living cells. Our focus here is on distinct ways of modeling the stiffness inherent in a chain molecule. We consider three types of stiffnesses-one yielding an energy penalty for local bends (energetic stiffness) and the other two forbidding certain classes of chain conformations (entropic stiffness). Using detailed Wang-Landau microcanonical Monte Carlo simulations, we study the interplay between the nature of the stiffness and the ground state conformation of a self-attracting chain. We find a wide range of ground state conformations, including a coil, a globule, a toroid, rods, helices, and zig-zag strands resembling β-sheets, as well as knotted conformations allowing us to bridge conventional polymer phases and biomolecular phases. An analytical mapping is derived between the persistence lengths stemming from energetic and entropic stiffness. Our study shows unambiguously that different stiffnesses play different physical roles and have very distinct effects on the nature of the ground state of the conformation of a chain, even if they lead to identical persistence lengths.

Phase Separation in Asymmetric Cell Division.

Asymmetric cell division (ACD) is a conserved strategy for achieving cell diversity. A cell can undergo an intrinsic ACD through asymmetric segregation of cell fate determinants or cellular organelles. Recently, a new biophysical concept known as biomolecular phase separation, through which proteins and/or RNAs autonomously form a highly concentrated non-membrane-enclosed compartment via multivalent interactions, has provided new insights into the assembly and regulation of many membrane-less or membrane-attached organelles. Intriguingly, biomolecular phase separation is suggested to drive asymmetric condensation of cell fate determinants during ACD as well as organization of cellular organelles involved in ACD. In this Perspective, I first summarize recent findings on the molecular basis governing intrinsic ACD. Then I will discuss how ACD might be regulated by formation of dense molecular assemblies via phase separation.

The Control Centers of Biomolecular Phase Separation: How Membrane Surfaces, PTMs, and Active Processes Regulate Condensation.

Biomolecular condensation is emerging as an essential process for cellular compartmentalization. The formation of biomolecular condensates can be driven by liquid-liquid phase separation, which arises from weak, multivalent interactions among proteins and nucleic acids. A substantial body of recent work has revealed that diverse cellular processes rely on biomolecular condensation and that aberrant phase separation may cause disease. Many proteins display an intrinsic propensity to undergo phase separation. However, the mechanisms by which cells regulate phase separation to build functional condensates at the appropriate time and location are only beginning to be understood. Here, we review three key cellular mechanisms that enable the control of biomolecular phase separation: membrane surfaces, post-translational modifications, and active processes. We discuss how these mechanisms may function in concert to provide robust control over biomolecular condensates and suggest new research avenues that will elucidate how cells build and maintain these key centers of cellular compartmentalization.

Interrogating the Structural Dynamics and Energetics of Biomolecular Systems with Pressure Modulation.

High hydrostatic pressure affects the structure, dynamics, and stability of biomolecular systems and is a key parameter in the context of the exploration of the origin and the physical limits of life. This review lays out the conceptual framework for exploring the conformational fluctuations, dynamical properties, and activity of biomolecular systems using pressure perturbation. Complementary pressure-jump relaxation studies are useful tools to study the kinetics and mechanisms of biomolecular phase transitions and structural transformations, such as membrane fusion or protein and nucleic acid folding. Finally, the advantages of using pressure to explore biomolecular assemblies and modulate enzymatic reactions are discussed.

Applications of gradient magnetic fields in studies of biological macromolecules

As a kind of physical environment, magnetic fields are widely used in many different fields, ranging from scientific research to industrial production. With the rapid development of magnetic technology, the importance of magnetic fields in scientific research and practical applications has become increasingly prominent. One notable research field is the application of magnetic fields in the research of biological macromolecules. In this research field, gradient magnetic fields, as one kind of magnetic fields, have attracted much attention and have been widely used in various research fields because they can induce effects caused not only by a magnetic field but also by a magnetic field gradient. This feature makes magnetic fields applicable in many research areas, except for those directly related to the magnetic field itself. So far, the application-based research of gradient magnetic fields involving biological macromolecules has mainly focused on the crystallization, separation and purification as well as the self-assembly of biological macromolecules. Since biological macromolecules usually need to complete various processes in their solution state, the influence of gradient magnetic fields on biological macromolecules is mainly realized through their influence on the solution system. It has been found that a gradient magnetic field can affect convection in biological macromolecular solutions through Lorentz forces and magnetization forces; in practice, partial or even complete suppression of convection can often be observed. In addition, other conditions or parameters of solutions are significantly affected by the gradient magnetic field. For example, in a gradient magnetic field, the following phenomena may occur: (1) superparamagnetic particles in the solution are strongly attracted by the magnetization force, and quick clustering of these particles can happen; (2) the solution itself may have some kind of solution structure; (3) the aggregation of biological macromolecules may show a preferred orientation in the magnetic field direction; (4) the diffusion coefficient of biological macromolecules in the solution will decrease; (5) the solutions viscosity will increase, and so on. As a result of these effects, the following phenomena can be observed: during the crystallization of biological macromolecules, a preferred orientation of the crystals and crystal quality improvement of the biological macromolecules can happen; in the process of separation and purification of biological macromolecules, high-purity and highly bioactive macromolecules can be obtained rapidly and efficiently; and, in the process of the self-assembly of biological macromolecules, abnormal behaviours can occur. Based on these specific effects, the utilization of gradient magnetic fields can be further explored in more professional applications. Currently, the most common applications include the utilization of gradient magnetic fields for obtaining high- quality protein crystals and the achievement of highly efficient and low-cost separation and purification of biological macromolecules. It is obvious that gradient magnetic fields have very important application value in biological macromolecular structure analysis technology, biological drug preparation and preservation technology, theoretical studies involving biomolecular phase separation and nucleation processes, and many other directions. In this paper, the fundamental effects of the gradient magnetic field on the solution of biological macromolecules are reviewed, the applications based on the effects are discussed, and the future prospects in this field are anticipated.

Why does biopolymer condensation matter?

Biomolecular condensation that occurs through phase separation is an emerging topic in cell biology. Ongoing research will continue to provide novel mechanistic insights into the involvement of biological condensates in a variety of normal and pathological cellular processes. In particular, a deeper understanding of the physical basis for biomolecular phase separation and of the properties of biopolymer condensates should emerge in the near future. Accordingly, connections between these processes and the organization of matter in living organisms will undergo fresh scrutiny. Biomolecules can phase separate and form condensates that have roles in diverse cellular processes and contexts. Michnick and Bergeron-Sandoval comment on this rapidly progressing field and envisage that the study of biological phase separation will bring new understanding of cell and developmental biology.

Biomolecular phases in transverse palatal distraction: A review.

Transverse palatal distraction is a biological process of regenerating new bone and enveloping soft tissues in the maxillary palate region. This technique is similar to Osteo-distraction (OD) procedure for bone lengthening in which gradual and controlled traction forces are applied on the osteotomy gaps to produce new bone in between the surgically separated bone segments. This review describes the different phases after osteotomy and the biological process involved during the new bone and soft tissue formation. The mechanical environment formed in the distraction area is due to the traction forces by the distractor appliance. This environment stimulates differentiation of pluripotent cells, neovascularization, osteogenesis and remodeling of newly formed bone. The role of different pro-inflammatory cytokines, interleukins, bone morphogenic proteins, transforming growth factors, fibroblast growth factors-2) and extracellular matrix proteins (osteonectin, osteopontin) during the distraction phases has been described in detail. Also, an important note on the nutritional aspect during Osteo-distraction will benefit the clinicians to guide their patients after osteotomy throughout the distraction process.

Coacervation in Biopolymers

In this review, a detailed discussion on salient features of intermolecular interactions leading to phase separation and coacervation is discussed. Biomolecular solutions exist as gels, coacervates and melts with each of these phases having its signature physico-chemical properties which is discussed in this review. The discussions are supported by robust experimental data obtained from an array of methods like turbidimetry, electrophoresis, viscosity, light scattering etc. The inevitability of the phenomenon of self-organization in biopolymers results in generation of a variety of soft matter phases which do not, however, make it predictable. For instance the associate aggregation is a process which remains obscure, as every protein aggregates in a different manner under different conditions. One known feature to the aggregation of proteins is the strong dependence upon pH, salt concentration, and temperature. Beyond the influence of these factors and their effects on aggregation, the process is not well understood. In summary, a comprehensive account of biomolecular phase states and their inherent attributes are presented in this review. Potential applications of coacervates are many starting from protein purification, drug encapsulation to treatment of organic plumes. This calls for better understanding of the coacervate structure and the transport of biomolecules inside this phase. Several questions pertaining to the structure of coacervates can arise. The foremost of these is, is it a gel-like or a solution-like phase? Though presence of hydrogen bonding and hydrophobic sites on the polyions influence biomolecular binding, they hardly play any role in deciding the persistence length of the polyion unlike the surface charge. Interestingly, we observed that the differential binding (SPB versus EB) was found to be a function of intrinsic persistence length only which we conclude as a significant observation.

Static and time-resolved synchrotron small-angle x-ray scattering studies of lyotropic lipidmesophases, model biomembranes and proteins in solution

In this work we discuss the use of small-angle x-ray scattering methods for investigating thetemperature and pressure dependent structure and phase behaviour of soft condensedmatter and in particular of biomolecular systems, such as lipid mesophases, modelbiomembrane systems as well as proteins in solution. In addition to temperature, pressurehas also been used as a physical parameter in these studies, in particular for studying theenergetics and phase behaviour of these systems, but also because high pressure is a featureof certain natural environments and because the high pressure phase behaviour ofbiomolecules is also of importance for biotechnological applications. By using thepressure jump relaxation technique in combination with time-resolved synchrotronsmall-angle x-ray scattering, the kinetics of biomolecular phase transitions canbe investigated. We applied the technique for studying lipid phase transitionsand protein unfolding/refolding reactions. After the discussion of the underlyingtheoretical concepts, several characteristic examples are presented and discussed.