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Amphiphilic proteins coassemble into multiphasic condensates
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and act as biomolecular surfactants
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Fleurie M. Kelley
1,a
, Bruna Favetta
1,b
, Roshan M. Regy
c
, Jeetain Mittal
c
, Benjamin S. Schuster
2,a
4
5
a
Department of Chemical and Biochemical Engineering, Rutgers University, Piscataway, NJ 08854
6
b
Department of Biomedical Engineering, Rutgers University, Piscataway, NJ 08854
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c
Artie McFerrin Department of Chemical Engineering, Texas A&M University, College Station, TX 77843
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1
These authors contributed equally to this work
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2
Corresponding author bss142@soe.rutgers.edu
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Abstract
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Cells contain membraneless compartments that assemble due to liquid-liquid phase separation,
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including biomolecular condensates with complex morphologies. For instance, certain condensates are
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surrounded by a film of distinct composition, such as Ape1 condensates coated by a layer of Atg19, required
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for selective autophagy in yeast. Other condensates are multiphasic, with nested liquid phases of distinct
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compositions and functions, such as in the case of ribosome biogenesis in the nucleolus. The size and
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structure of such condensates must be regulated for proper biological function. We leveraged a bio-inspired
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approach to discover how amphiphilic, surfactant-like proteins may contribute to the structure and size
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regulation of biomolecular condensates. We designed and examined families of amphiphilic proteins
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comprising one phase-separating domain and one non-phase separating domain. In particular, these
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proteins contain the soluble structured domain glutathione S-transferase (GST) or maltose binding protein
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(MBP), fused to the intrinsically disordered RGG domain from P granule protein LAF-1. When one
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amphiphilic protein is mixed in vitro with RGG-RGG, the proteins assemble into enveloped condensates,
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with RGG-RGG at the core, and the amphiphilic protein forming the surface film layer. Importantly, we found
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that MBP-based amphiphiles are surfactants and control droplet size, with increasing surfactant
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concentration resulting in smaller droplet radii. In contrast, GST-based amphiphiles at increased
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concentrations co-assemble with RGG-RGG into multiphasic structures. We propose a mechanism for
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these experimental observations, supported by molecular simulations of a minimalist model. We speculate
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that surfactant proteins may play a significant role in regulating the structure and function of biomolecular
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condensates.
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2
Introduction
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The intracellular environment is like a complex emulsion. This paradigm originated more than a
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century ago but is enjoying a renaissance, with recent discoveries revealing the important role of liquid-
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liquid phase separation (LLPS) in biology
1–3
. LLPS of proteins and nucleic acids underlies the formation of
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membraneless organelles, alternatively called biomolecular condensates, which are distinct intracellular
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compartments that lack a delimiting membrane
2,3
. Biomolecular condensates contribute to numerous cell
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functions, including stress response, gene regulation, and signaling
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. Conversely, aberrant phase
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separation due to mutations and age-related processes is implicated in diseases such as
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neurodegeneration and cancer
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. Deciphering the rules of self-assembly of biomolecular condensates has
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therefore emerged as a promising avenue for elucidating fundamental principles of biological structure,
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function, and dysfunction.
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Despite significant recent progress in understanding the biophysics of biomolecular condensates,
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many open questions remain
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. One key question is what molecular phenomena govern the spontaneous
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assembly of condensates with core-shell or multiphasic structures. Another important question is how cells
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tune the size of biomolecular condensates. Here, we sought to gain insight into both questions by examining
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how amphiphilic, surfactant-like proteins contribute to the self-assembly and regulation of biomolecular
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condensates. Amphiphiles are typically defined as molecules comprising separate hydrophilic and
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hydrophobic parts. Here, we note the etymology (in Greek, “amphi” means both and “philia” means
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friendship or love) and use the term amphiphile to describe proteins comprising one domain that has affinity
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for biomolecular condensates and one domain that has affinity for the dilute phase.
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Surfactants are substances, generally amphiphiles, that adsorb to interfaces and decrease
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interfacial tension. Extracellularly, pulmonary surfactant lining the alveoli plays a vital role in lung physiology
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by reducing the work of breathing
7,8
. However, the role of surfactants in the emulsion-like intracellular milieu
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is just beginning to be explored
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. In biological systems, the surfactant-like protein Ki-67 prevents individual
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chromosomes from coalescing during early stages of mitosis by forming a repulsive molecular brush layer
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10
. Some biomolecular condensates are reminiscent of surfactant-laden emulsions, although their physical
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chemistry remains to be elucidated. For instance, Atg19 forms a thin surface layer surrounding Ape1
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condensates that is necessary for selective autophagy of Ape1 in yeast
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. Inspired by such examples, we
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was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted May 29, 2021. ; https://doi.org/10.1101/2021.05.28.446223doi: bioRxiv preprint
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hypothesized that a minimal system comprising surfactant-like proteins interacting with phase-separating
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proteins could recapitulate enveloped condensate structures observed in nature.
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Moreover, condensates exhibit a variety of multiphase and multilayer structures underpinning their
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biological functions. Bre1 assembles as a shell surrounding Lge1 condensates, generating a catalytic
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condensate that functions to accelerate ubiquitination of histone H2B in yeast
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. The nucleolus is comprised
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of coexisting liquid phases of differing interfacial tensions
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, while P granules contain coexisting liquid and
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gel phases
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. Stress granules
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, nuclear speckles
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, paraspeckles
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, and reconstituted polypeptide/RNA
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complex coacervates also exhibit core-shell structures sensitive to stoichiometry and competitive binding
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18–21
. Functionally related condensates can remain in contact without coalescing, as in the case of stress
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granules and P-bodies
22
, or P granules and Z granules
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. We asked whether amphiphilic proteins could
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contribute to the complex morphologies of biomolecular condensates that have been observed within cells,
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just as synthetic amphiphiles and surfactant systems exhibit rich structures and phase behaviors
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.
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Surfactant-like proteins could have additional important functional consequences, including but not
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limited to modulating biomolecular condensate size, which in turn influences biochemical processes
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through condensate size-dependent effects on molecular concentrations and diffusion
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. Biomolecular
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condensates are often observed in cells as multiple smaller droplets, rather than as a single larger droplet,
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even though the latter is expected to be thermodynamically favored. Recent studies have attributed the
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apparent metastability of biomolecular condensates in various contexts to surface charge
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, cytoskeletal
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caging
28,29
, membrane association
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, and exhaustion of available binding sites
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; active processes can
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also maintain the emulsified, multi-droplet state in vivo
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. An additional possibility, which we examine here,
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is that surfactant proteins may help stabilize biomolecular condensates.
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To address these questions, we adopted a bottom-up, bioinspired approach, seeking to leverage
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a simplified system to shed light on the role of amphiphilic proteins in the self-assembly of biomolecular
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condensates. We designed amphiphilic proteins containing an intrinsically disordered region (IDR) fused to
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folded domains. The IDR is a phase-separating domain, whereas the folded domains are not. When one of
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these amphiphilic fusion proteins is mixed at low concentrations with the IDR alone, the two proteins
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assemble such that the amphiphilic protein forms a film that coats the IDR core. We demonstrate several
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extensions of this observation, including the important finding that condensate size can be controlled by
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was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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4
varying surfactant protein concentration. Furthermore, when amphiphilic proteins with different folded
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domains are mixed together with the core IDR, we observe competition between different surfactant
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proteins for binding to the condensate interface. Interestingly, one family of amphiphilic proteins exhibits
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varied morphologies, including multiphasic condensates, and we map the rich concentration-dependent
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phase behavior. To gain mechanistic insight into these experimental observations, we present a
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minimalistic computational model that recapitulates the range of behaviors observed experimentally by
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varying the strength of interaction between domains. Our experiments and simulations suggest that
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amphiphile-condensate assembly is determined by the strength of interaction between the amphiphile and
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the IDR core, as well as interactions between the folded domain of the amphiphile. Taken together, this
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work illustrates the diverse interfacial phenomena that can arise from interactions between condensates
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and amphiphilic proteins, notably raising the possibility that surfactant proteins may play a significant role
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in regulating the structure and function of biomolecular condensates.
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Results
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Designing Amphiphilic Proteins to Act as Biological Surfactants
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In prior work, we examined the partitioning of client proteins into biomolecular condensates
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. We
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focused on the RGG domain from LAF-1, a prototypical arginine/glycine-rich intrinsically disordered protein
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(IDP) involved in P granule assembly in C. elegans
34–36
. Tandem repeats of the RGG domain (RGG-RGG)
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phase separate in vitro at concentrations >1 µM under physiological conditions. We found that RFP and
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GST-RFP are excluded from RGG-RGG condensates; RFP denotes red fluorescent protein and GST
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denotes glutathione S-transferase, which is widely used as a solubility-enhancing affinity tag in recombinant
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protein production
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. In contrast, we observed that RFP fused to one or two RGG domains partitioned into
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RGG-RGG condensates. These results demonstrated that client partitioning into or exclusion from RGG-
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RGG condensates depends on the balance between the “RGG-philic” and “RGG-phobic” content of the
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client protein. Here, we hypothesized that a third, intermediate outcome is possible: that with the right
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balance, a client protein may localize to the interface between the condensate and dilute phases, coating
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the condensate and displaying surfactant-like behavior.
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We therefore asked whether we could engineer amphiphilic proteins that assemble as a film on the
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was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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surface of RGG-RGG condensates. We designed a family of amphiphilic fusion proteins containing a
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phase-separating domain (RGG-philic) fused to a non-phase separating domain (RGG-phobic) (SI
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Appendix Fig. 1). Two representative protein constructs from this family are MBP-GFP-RGG and GST-
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GFP-RGG (Fig. 1A). MBP denotes maltose binding protein, which like GST is a well-known affinity tag that
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enhances protein solubility
37–40
. GFP denotes enhanced green fluorescent protein with the monomerizing
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A206K mutation. By combining RGG, GST, MBP, and fluorescent protein domains, our aim was to generate
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amphiphilic fusion proteins with RGG-philic and RGG-phobic parts. We hypothesized that upon mixing the
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amphiphilic proteins with RGG-RGG, the poorly soluble RGG domain in the amphiphiles would interact with
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and orient towards the inner RGG-RGG condensate phase, while the RGG-phobic MBP and GST domains
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would interact with and orient towards the outer dilute phase (Fig. 1B).
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Figure 1: Amphiphilic proteins MBP-GFP-RGG and GST-GFP-RGG form a film around RGG-RGG.
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A, Schematic domain diagram for MBP-GFP-RGG, GST-GFP-RGG, and RGG-RGG. Length is
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proportional to number of base pairs. B, Schematic model of the interaction between amphiphilic proteins
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and RGG-RGG. The amphiphilic protein is depicted at the interface between the two phases, surrounding
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the RGG-RGG core, with the phase-separating RGG domain facing the RGG-RGG core and the non-
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phase-separating GST or MBP domain facing the aqueous phase. C, Transmitted light and fluorescence
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imaging of the film formed by mixing RGG-RGG and MBP-GFP-RGG or GST-GFP-RGG in a 10:1
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concentration ratio (10 μM RGG-RGG, 1 μM MBP-GFP-RGG or GST-GFP-RGG). For this and
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subsequent figures, buffer was 150 mM NaCl, 20 mM Tris, pH 7.5. Right: Fluorescence intensities were
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quantified as line profiles across individual condensates, which were normalized and averaged. n, number
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of condensates. Scale bars, 5 μm.
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Amphiphilic Proteins Coat Biomolecular Condensates, Resembling Surfactants
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To test our hypothesis, we mixed RGG-RGG with the amphiphilic proteins GST-GFP-RGG or MBP-
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GFP-RGG in physiological buffer and used microscopy to observe the resulting assemblies. In these binary
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