Frustrated Microphase Separation Produces Interfacial Environment within Biological Condensates

Reviewer #1 (Public Review):

This is an interesting, informative, and well-designed study that combines theoretical and experimental methodologies to tackle the phenomenon of higher-resolution structures/substructures in model biomolecular condensates. The results should be published. However, there is significant room for improvement in the presentation and interpretation of the results. As it stands, the precise definition of "frustration," which is a main theme of this manuscript (as emphasized in the title), is not sufficiently well articulated. This situation should be rectified to avoid "frustration" becoming a "catch-all" term without a clear perimeter of applicability rather than a precise, informative description of the physical state of affairs. There are also a few other concerns, e.g., regarding interpretation of correlation of phase-separation critical temperature and transfer free energy of amino acid residues as well as the difference between critical temperature and onset temperature, and the way the simulated configurations are similar to that of gyroids. Accordingly, the manuscript should be revised to address the following:

1. It is accurately pointed out on p.4 that elastin-like polypeptides (ELPs) undergo heat-induced phase separation and therefore exhibit lower critical solution temperatures (LCSTs). But it is not entirely clear how this feature is reproduced by the authors' simulation. A relationship between simulated surface tension and "transition temperature" is provided in Fig.1C; but is the "transition temperature" (authors cited ref.41 by Urry) the same as critical temperature? Apparently, Urry's Tt is "critical onset temperature", the temperature when phase separation happens at a given polymer concentration. This is different from the (global) critical temperature LCST - though the two may be correlated-or not-depending on the shape of the phase boundary. Moreover, is the MOFF coarse-grained forcefield (first step in the multi-scale simulation), by itself, capable of reproducing heat-induced phase separation in a way similar to the forcefield of Dignon et al., ACS Cent Sci 5, 821-230 (2019)? Or is this temperature-dependent effect appearing only subsequently, after the implementation of the MARTINI and/or all-atom steps? Clarification is needed. To afford a more informative context for the authors' introductory discussion, the aforementioned Dignon et al. work and the review by Cinar et al. [Chem Eur J 25, 13049-13069 (2019)], both touching upon the physical underpinning of the LCST feature of elastin, should also be cited along with refs.41-43.

2. "Frustration" and "frustrated" are used prominently in the manuscript to characterize certain observed molecular configurations (11 times total, in both the title and in the abstract). Apparently, it is the most significant conceptual pronouncement of this work, hence its precise meaning is of central importance to the authors' thesis. Whereas one should recognize that the theoretical and experimental observations are striking without invocation of the "frustration" terminology, usage of the term can be useful if it offers a unifying conceptual framework. However, as it stands, a clear definition of the term "frustration" is lacking, leaving readers to wonder what molecular configurations are considered "frustrated" and what are not (i.e., is the claim of observation of frustration falsifiable?). For instance, "frustrated microphase separation" appears in both the title and abstract. A logical question one may ask is: "Are all microphase separations frustrated"? If the answer is in the affirmative, does invocation of the term "frustration" add anything to our physical insight? If the answer is not in the affirmative, then how does one distinguish between microphase separations that are frustrated from those that are not frustrated? Presumably all simulated and experimental molecular configurations in the present study are those of lowest free energy for the given temperature. In other words, they are what they are. In the discussion about frustrated phase separation on p.13, for example, the authors appear to refer to the fact that chain connectivity is preventing hydrophobic residues to come together in a way to achieve the most favorable interactions as if there were no chain connectivity (one may imagine in that case all the hydrophobic residues will form a large cluster without microphase separation). Is this what the authors mean by "frustration"? If that's true, isn't that merely stating the obvious, at least for the observed microphase separation? In general, does "frustration" always mean deviation of actual, physical molecular configurations from certain imagined/hypothetical/reference molecular configurations, and therefore dependent upon the choice of the imagined reference configuration? If this is how the authors apply the term "frustration" in the present work, what is the zero-frustration reference state/configuration for microphase separation? And, similarly, what is the zero-frustration reference state/configuration when frustrated EPS-water interactions are discussed (~p.14-p.15, Fig.5)? How do non-frustrated water-protein interactions look like? Is the classic clathrate-like organization of water hydrogen bonds around small nonpolar solute "frustrated"?

3. In the discussion about the correlation of various transfer free energy scales for amino acids and Urry's critical onset temperature (ref.41) on p.11 and Fig.4, is there any theoretical relationship to be expected between the interactions among amino acids of ELPs and their critical onset temperatures? While a certain correlation may be intuitively expected if the free energy scale "is working", is there any theoretical insight into the mathematical form of this relationship? A clarifying discussion is needed because it bears logically on whether the observed correlation or lack thereof for different transfer energy scales is a good indication of the adequacy of the energy scales in describing the actual physical interactions at play. This question requires some prior knowledge of the expected mathematical relationship between interaction parameters and onset temperature.

4. To provide a more comprehensive context for the present study, it is useful to compare the microphase separation seen in the authors' simulation with the micelle-like structures observed in recent simulated condensed/aggregated states of hydrophobic-polar (HP) model sequences in Statt et al., J Chem Phys 152, 075101 (2020) [see esp. Fig.6] and Wessén et al., J Phys Chem B 126, 9222-9245 (2022) [see, e.g., Fig.10].

5. "Gyroid-like morphology" is mentioned several times in the manuscript (p.4, p.8, p.17, Fig.S3). This is apparently an interesting observation, but a clear explanation is lacking. A more detailed and specific discussion, perhaps with additional graphical presentations, should be provided to demonstrate why the simulated condensed-phase ELP configurations are similar to the classical description of gyroid as in, e.g., Terrones & Mackay, Chem Phys Lett 207, 45-50 (1993) and Lambert et al., Phil Trans R Soc A 354, 2009-2023 (1996).

Reviewer #2 (Public Review):

Summary:
Latham A.P. et al. apply simulations and FLIM to analyse several di-block elastin-like polypetides and connect their sequence to the micro-structure of coacervates resulting from their phase-separation.

Strengths:
Understanding the molecular grammar of phase separating proteins and the connection with mesoscale properties of the coacervates is highly relevant. This work provides insights into micro-structures of coacervates resulting from di-block polypetides.

Weaknesses:
The results apply to a very specific architecture (di-block polypetides) with specific sequences.