Sequence-encoded Spatiotemporal Dependence of Viscoelasticity of Protein Condensates Using Computational Microrheology.

Many biomolecular condensates act as viscoelastic complex fluids with distinct cellular functions. Deciphering the viscoelastic behavior of biomolecular condensates can provide insights into their spatiotemporal organization and physiological roles within cells. Though there is significant interest in defining the role of condensate dynamics and rheology in physiological functions, the quantification of their time-dependent viscoelastic properties is limited and mostly done through experimental rheological methods. Here, we demonstrate that a computational passive probe microrheology technique, coupled with continuum mechanics, can accurately characterize the linear viscoelasticity of condensates formed by intrinsically disordered proteins (IDPs). Using a transferable coarse-grained protein model, we first provide a physical basis for choosing optimal values that define the attributes of the probe particle, namely its size and interaction strength with the residues in an IDP chain. We show that the technique captures the sequence-dependent viscoelasticity of heteropolymeric IDPs that differ either in sequence charge patterning or sequence hydrophobicity. We also illustrate the technique's potential in quantifying the spatial dependence of viscoelasticity in heterogeneous IDP condensates. The computational microrheology technique has important implications for investigating the time-dependent rheology of complex biomolecular architectures, resulting in the sequence-rheology-function relationship for condensates.

Sequence-Dependent Conformational Transitions of Disordered Proteins During Condensation.

Intrinsically disordered proteins (IDPs) can form biomolecular condensates through phase separation. It is recognized that the conformation of IDPs in the dense and dilute phases as well as at the interfaces of condensates can critically impact the resulting properties associated with their functionality. However, a comprehensive understanding of the conformational transitions of IDPs during condensation remains elusive. In this study, we employ a coarse-grained polyampholyte model, comprising an equal number of oppositely charged residues-glutamic acid and lysine-whereby conformations and phase behavior can be readily tuned by altering the protein sequence. By manipulating the sequence patterns from perfectly alternating to block-like, we obtain chains with ideal-like conformations to semi-compact structures in the dilute phase, while in the dense phase, the chain conformation is approximately that of an ideal chain, irrespective of the protein sequence. By performing simulations at different concentrations, we find that the chains assemble from the dilute phase through small oligomeric clusters to the dense phase, accompanied by a gradual swelling of the individual chains. We further demonstrate that these findings are applicable to several naturally occurring proteins involved in the formation of biological condensates. Concurrently, we delve deeper into the chain conformations within the condensate, revealing that chains at the interface show a strong sequence dependence, but remain more collapsed than those in the bulk-like dense phase. This study addresses critical gaps in our knowledge of IDP conformations within condensates as a function of protein sequence.

Sequence-Dependent Material Properties of Biomolecular Codensates and their Relation to Dilute Phase Conformations.

Material properties of phase-separated biomolecular assemblies, enriched with disordered proteins, dictate their ability to participate in many cellular functions. Despite the significant effort dedicated to understanding how the sequence of the disordered protein drives its phase separation to form condensates, little is known about the sequence determinants of condensate material properties. Here, we computationally decipher these relationships for charged disordered proteins using model sequences comprised of glutamic acid and lysine residues as well as naturally occurring sequences of LAF1's RGG domain and DDX4's N-terminal domain. We do so by delineating how the arrangement of oppositely charged residues within these sequences influences the dynamical, rheological, and interfacial properties of the condensed phase through equilibrium and non-equilibrium molecular simulations using the hydropathy scale and Martini models. Our computations yield material properties that are quantitatively comparable with experimentally characterized condensate systems. Interestingly, we find that the material properties of both the model and natural proteins respond similarly to the segregation of charges, despite their very different sequence compositions. Condensates of the highly charge-segregated sequences exhibit slower dynamics than the uniformly charge-patterned sequences, because of their comparatively long-lived molecular contacts between oppositely charged residues. Surprisingly, the molecular interactions within the condensate are highly similar to those within a single-chain for all sequences. Consequently, the condensate material properties of charged disordered proteins are strongly correlated with their dense phase contact dynamics and their single-chain structural properties. Our findings 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.

Conformational Properties of Polymers at Droplet Interfaces as Model Systems for Disordered Proteins.

Polymer models serve as useful tools for studying the formation and physical properties of biomolecular condensates. In recent years, the interface dividing the dense and dilute phases of condensates has been discovered to be closely related to their functionality, but the conformational preferences of the constituent proteins remain unclear. To elucidate this, we perform molecular simulations of a droplet formed by phase separation of homopolymers as a surrogate model for the prion-like low-complexity domains. By systematically analyzing the polymer conformations at different locations in the droplet, we find that the chains become compact at the droplet interface compared with the droplet interior. Further, segmental analysis revealed that the end sections of the chains are enriched at the interface to maximize conformational entropy and are more expanded than the middle sections of the chains. We find that the majority of chain segments lie tangential to the droplet surface, and only the chain ends tend to align perpendicular to the interface. These trends also hold for the natural proteins FUS LC and LAF-1 RGG, which exhibit more compact chain conformations at the interface compared to the droplet interior. Our findings provide important insights into the interfacial properties of biomolecular condensates and highlight the value of using simple polymer physics models to understand the underlying mechanisms.

Effect of Charge Distribution on the Dynamics of Polyampholytic Disordered Proteins.

The stability and physiological function of many biomolecular coacervates depend on the structure and dynamics of intrinsically disordered proteins (IDPs) that typically contain a significant fraction of charged residues. Although the effect of relative arrangement of charged residues on IDP conformation is a well-studied problem, the associated changes in dynamics are far less understood. In this work, we systematically interrogate the effects of charge distribution on the chain-level and segmental dynamics of polyampholytic IDPs in dilute solutions. We study a coarse-grained model polyampholyte consisting of an equal fraction of two oppositely charged residues (glutamic acid and lysine) that undergoes a transition from an ideal chain-like conformation for uniformly charge-patterned sequences to a semi-compact conformation for highly charge-segregated sequences. Changes in the chain-level dynamics with increasing charge segregation correlate with changes in conformation. The chain-level and segmental dynamics conform to simple homopolymer models for uniformly charge-patterned sequences but deviate with increasing charge segregation, both in the presence and absence of hydrodynamic interactions. We discuss the significance of these findings, obtained for a model polyampholyte, in the context of a charge-rich intrinsically disordered region of the naturally occurring protein LAF-1. Our findings have important implications for understanding the effects of charge patterning on the dynamics of polyampholytic IDPs in dilute conditions using polymer scaling theories.