Intrinsically Disordered Landscapes for Human CD4 Receptor Peptide.

Because of their inherent structural plasticity, intrinsically disordered proteins (IDPs) are generally difficult to characterize, both experimentally and via simulations. In this work, an approach for studying IDPs within the potential energy landscape framework is implemented and tested. Specifically, human CD4 receptor peptide, a disordered region implicated in HIV-1 infection, is characterized via basin-hopping parallel tempering and discrete path sampling. We also investigate the effects of three state-of-the-art AMBER force fields (ff99SB-ILDN, ff14ipq, and ff14SB) on the energy landscape. The results for ff99SB-ILDN exhibit the best agreement with experiment. Metastable states identified on the free energy surface help to unify, and are consistent with, several earlier predictions, and may serve as starting points for probing the reaction interface between CD4 and HIV-1 accessory proteins.epsr

Energy Landscape for Fold-Switching in Regulatory Protein RfaH.

The C-terminal domain (CTD) of bacterial regulatory protein RfaH undergoes a dramatic structural rearrangement from an α-helical hairpin to a β-barrel. We employ a quasi-continuous interpolation scheme and geometry optimization techniques to construct a kinetic transition network for this process. The computed free energy landscape at 310 K is multifunneled, and the predicted free energy ensembles are in good agreement with experiment and other simulation studies. We find that rearrangement from the α-helical conformer to the β-sheet proceeds via an essentially unstructured state. The techniques refined for the present system should be transferable to other protein conformational switches, with the potential to advance our understanding of such systems.epsr

Valency and Binding Affinity Variations Can Regulate the Multilayered Organization of Protein Condensates with Many Components.

Biomolecular condensates, which assemble via the process of liquid-liquid phase separation (LLPS), are multicomponent compartments found ubiquitously inside cells. Experiments and simulations have shown that biomolecular condensates with many components can exhibit multilayered organizations. Using a minimal coarse-grained model for interacting multivalent proteins, we investigate the thermodynamic parameters governing the formation of multilayered condensates through changes in protein valency and binding affinity. We focus on multicomponent condensates formed by scaffold proteins (high-valency proteins that can phase separate on their own via homotypic interactions) and clients (proteins recruited to condensates via heterotypic scaffold-client interactions). We demonstrate that higher valency species are sequestered to the center of the multicomponent condensates, while lower valency proteins cluster towards the condensate interface. Such multilayered condensate architecture maximizes the density of LLPS-stabilizing molecular interactions, while simultaneously reducing the surface tension of the condensates. In addition, multilayered condensates exhibit rapid exchanges of low valency proteins in and out, while keeping higher valency proteins-the key biomolecules involved in condensate nucleation-mostly within. We also demonstrate how modulating the binding affinities among the different proteins in a multicomponent condensate can significantly transform its multilayered structure, and even trigger fission of a condensate into multiple droplets with different compositions.Engineering and Physical Sciences Research Council (EPSRC) scholarship to Ignacio Sanchez-Burgo

RNA length has a non-trivial effect in the stability of biomolecular condensates formed by RNA-binding proteins.

Funder: Oppenheimer FellowshipFunder: Roger Ekins FellowshipFunder: Derek Brewer Emmanuel College scholarshipBiomolecular condensates formed via liquid-liquid phase separation (LLPS) play a crucial role in the spatiotemporal organization of the cell material. Nucleic acids can act as critical modulators in the stability of these protein condensates. To unveil the role of RNA length in regulating the stability of RNA binding protein (RBP) condensates, we present a multiscale computational strategy that exploits the advantages of a sequence-dependent coarse-grained representation of proteins and a minimal coarse-grained model wherein proteins are described as patchy colloids. We find that for a constant nucleotide/protein ratio, the protein fused in sarcoma (FUS), which can phase separate on its own-i.e., via homotypic interactions-only exhibits a mild dependency on the RNA strand length. In contrast, the 25-repeat proline-arginine peptide (PR25), which does not undergo LLPS on its own at physiological conditions but instead exhibits complex coacervation with RNA-i.e., via heterotypic interactions-shows a strong dependence on the length of the RNA strands. Our minimal patchy particle simulations suggest that the strikingly different effect of RNA length on homotypic LLPS versus RBP-RNA complex coacervation is general. Phase separation is RNA-length dependent whenever the relative contribution of heterotypic interactions sustaining LLPS is comparable or higher than those stemming from protein homotypic interactions. Taken together, our results contribute to illuminate the intricate physicochemical mechanisms that influence the stability of RBP condensates through RNA inclusion

Size conservation emerges spontaneously in biomolecular condensates formed by scaffolds and surfactant clients

Funder: Junior Research Fellow at Kings CollegeFunder: Ernest Oppenheimer Memorial Trust; doi: http://dx.doi.org/10.13039/501100009978Abstract: Biomolecular condensates are liquid-like membraneless compartments that contribute to the spatiotemporal organization of proteins, RNA, and other biomolecules inside cells. Some membraneless compartments, such as nucleoli, are dispersed as different condensates that do not grow beyond a certain size, or do not present coalescence over time. In this work, using a minimal protein model, we show that phase separation of binary mixtures of scaffolds and low-valency clients that can act as surfactants—i.e., that significantly reduce the droplet surface tension—can yield either a single drop or multiple droplets that conserve their sizes on long timescales (herein ‘multidroplet size-conserved’ scenario’), depending on the scaffold to client ratio. Our simulations demonstrate that protein connectivity and condensate surface tension regulate the balance between these two scenarios. The multidroplet size-conserved scenario spontaneously arises at increasing surfactant-to-scaffold concentrations, when the interfacial penalty for creating small liquid droplets is sufficiently reduced by the surfactant proteins that are preferentially located at the interface. In contrast, low surfactant-to-scaffold concentrations enable continuous growth and fusion of droplets without restrictions. Overall, our work proposes one thermodynamic mechanism to help rationalize how size-conserved coexisting condensates can persist inside cells—shedding light on the roles of protein connectivity, binding affinity, and droplet composition in this process

Research data supporting - Energy landscapes and heat capacity signatures for peptides correlate with phase separation propensity

The discrete path sampling databases for various hexapeptides obtained using AMBER interfaced with GMIN, OPTIM and PATHSAMPLE programs for energy landscape exploration. Detailed description is given in the README file.This work was supported by Engineering and Physical Sciences Research Council (EPSRC) (D.J.W, grant number EP/N035003/1); the Cambridge Commonwealth, European and International Trust; the Allen, Meek and Read Fund; the Santander fund, St Edmund’s College, University of Cambridge; and the Trinity-Henry Barlow Honorary Award (Nicy)

Aromatic and arginine content drives multiphasic condensation of protein-RNA mixtures.

Multiphasic architectures are found ubiquitously in biomolecular condensates and are thought to have important implications for the organization of multiple chemical reactions within the same compartment. Many of these multiphasic condensates contain RNA in addition to proteins. Here, we investigate the importance of different interactions in multiphasic condensates comprising two different proteins and RNA using computer simulations with a residue-resolution coarse-grained model of proteins and RNA. We find that in multilayered condensates containing RNA in both phases, protein-RNA interactions dominate, with aromatic residues and arginine forming the key stabilizing interactions. The total aromatic and arginine content of the two proteins must be appreciably different for distinct phases to form, and we show that this difference increases as the system is driven toward greater multiphasicity. Using the trends observed in the different interaction energies of this system, we demonstrate that we can also construct multilayered condensates with RNA preferentially concentrated in one phase. The "rules" identified can thus enable the design of synthetic multiphasic condensates to facilitate further study of their organization and function.We acknowledge funding from the University of Cambridge Ernest Oppenheimer Fund [PYC], the Winton Programme for the Physics of Sustainability [PYC, RC-G], the European Research Council under the European Union's Horizon 2020 research and innovation programme [grant 803326; RC-G]. JAJ was a Junior Research Fellow at King's College when this work was undertaken. This work was performed using resources provided by the Cambridge Tier-2 system operated by the University of Cambridge Research Computing Service funded by EPSRC Tier-2 capital grant EP/P020259/1 [RC-G, JAJ, AR], and the ARCHER2 UK National Supercomputing Service via the UK High-End Computing Consortium for Biomolecular Simulation (HEC BioSim) supported by EPSRC grant EP/R029407/1

Energy landscapes and heat capacity signatures for peptides correlate with phase separation propensity

Phase separation plays an important role in the formation of membraneless compart- ments within the cell, and intrinsically disordered proteins with low-complexity se- quences can drive this compartmentalisation. Various intermolecular forces, such as aromatic–aromatic and cation–aromatic interactions, promote phase separation. How- ever, little is known about how the ability of proteins to phase separate under physiolog- ical conditions is encoded in their energy landscapes, and this is the focus of the present investigation. Our results provide a first glimpse into how the energy landscapes of min- imal peptides that contain π–π and cation–π interactions differ from the peptides that lack amino acids with such interactions. The peaks in the heat capacity (CV ) as a function of temperature report on alternative low-lying conformations that differ significantly in terms of their enthalpic and entropic contributions. The CV analysis and subsequent quan- tification of frustration of the energy landscape suggest that the interactions that promote phase separation leads to features (peaks or inflection points) at low temperatures in CV , more features may occur for peptides containing residues with better phase separation propensity and the energy landscape is more frustrated for such peptides. Overall, this work links the features in the underlying single-molecule potential energy landscapes to their collective phase separation behaviour, and identifies quantities (CV and frustration metric) that can be utilised in soft material design

Thermodynamic origins of two-component multiphase condensates of proteins.

Acknowledgements: We acknowledge funding from the University of Cambridge Ernest Oppenheimer Fund [PYC], the Winton Programme for the Physics of Sustainability [PYC, RC-G], the European Research Council under the European Union's Horizon 2020 research and innovation programme [grant 803326; RC-G]. JAJ is a Junior Research Fellow at King's College. This work was performed using resources provided by the Cambridge Tier-2 system operated by the University of Cambridge Research Computing Service funded by EPSRC Tier-2 capital grant EP/P020259/1 [RC-G, JAJ, AR]. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.Intracellular condensates are highly multi-component systems in which complex phase behaviour can ensue, including the formation of architectures comprising multiple immiscible condensed phases. Relying solely on physical intuition to manipulate such condensates is difficult because of the complexity of their composition, and systematically learning the underlying rules experimentally would be extremely costly. We address this challenge by developing a computational approach to design pairs of protein sequences that result in well-separated multilayered condensates and elucidate the molecular origins of these compartments. Our method couples a genetic algorithm to a residue-resolution coarse-grained protein model. We demonstrate that we can design protein partners to form multiphase condensates containing naturally occurring proteins, such as the low-complexity domain of hnRNPA1 and its mutants, and show how homo- and heterotypic interactions must differ between proteins to result in multiphasicity. We also show that in some cases the specific pattern of amino-acid residues plays an important role. Our findings have wide-ranging implications for understanding and controlling the organisation, functions and material properties of biomolecular condensates.We acknowledge funding from the University of Cambridge Ernest Oppenheimer Fund [PYC], the Winton Programme for the Physics of Sustainability [PYC, RC-G], the European Research Council under the European Union’s Horizon 2020 research and innovation programme [grant 803326; RC-G]. JAJ is a Junior Research Fellow at King’s College. This work was performed using resources provided by the Cambridge Tier-2 system operated by the University of Cambridge Research Computing Service funded by EPSRC Tier-2 capital grant EP/P020259/1 [RC-G, JAJ, AR]