Disordered proteins: the next frontier in therapeutics?

 

Howard Stone at Princeton, Ozgur Sahin at Columbia, and their colleagues have written a wonderfully imaginative review of what they call “hydration solids” (Nature 619, 500; 2023 - here). By this I don’t mean to imply anything fantastical about it – I simply like this way of framing the notion that hydration water can play an important structural and mechanical role in some hygroscopic biological materials. The idea here is that the mechanical properties are governed by the hydration forces operative within a fluid-filled porous elastic medium. The notion is motivated and explored by studying the mechanical behaviour of bacterial spores, but the same principles might apply to wood, pollen grains, keratinous materials and silk. “Such ‘hydration solids’, which can exchange their essential constituent water with the environment and have it flow through the material, are potentially abundant in the environment”, they write.

 

There is a fascinating paper in PRL from Chunyi Zhang (Mike Klein’s group) at Temple University in Philadelphia that investigates why the dielectric permittivity of salt water can actually decrease as more salt is added (C. Zhang et al., Phys. Rev. Lett. 131, 076801; 2023 - here). Using a deep neural network trained on the results of density functional theory, the authors show that this is not some kind of saturation effect but arises because of the way the ionic hydration shells disrupt the hydrogen-bonded network of the water and thereby suppress the collective response to electric fields.

 

It has been recognized at least since the early 1970s (and explored by the late, great Jack Dunitz) that changes in enthalpy and in entropy of associations between biomolecules (such as receptor-ligand pairings), due for example to small changes in molecular structure, seem often to compensate for one another so as to entail little change in the Gibbs free energy of binding. Why this is so has been much debated. An intuitive explanation is that a more favourable enthalpic contribution to binding creates a corresponding decrease in conformational freedom and thus a loss in entropy. But consideration of that balance must also take into account changes in hydration due to reorganization of the local hydrogen-bonded network. The Whitesides group (Breiten et al., JACS  135, 15579; 2013) has argued that water reorganization is in fact the key source of enthalpy-entropy compensation. That idea is examined, and ultimately supported, in a study by Shensheng Chen and Zhen-Gang Wang at Caltech (J. Phys. Chem. B 127, 6825; 2023 - here), using MD simulations of model charged polymers. They find that the hydrophobic interactions resulting from reorganization of hydration water show temperature dependencies that can account for the close correlation between the deltaH and TdeltaS terms. The effects of electrostatic interactions and polymer conformational changes are, in comparison, minor. Water is, it seems, in control.

Liquid-liquid phase separation (LLPS) has become a vibrant topic in cell biology now that it’s clear cells make use of it in a variety of ways and circumstances for partitioning and sequestering biomolecules for purposes ranging from gene regulation to RNA splicing to stress responses. The globular droplets – condensates – formed in this process have a higher density than the surrounding cell fluid, but it’s still not really understood what characteristics of biomolecules promote this new phase. That understanding could be useful for being able to control the phase separation process for possible therapeutic purposes – or indeed for designing peptides to prevent pathogenic aggregation. The condensates are not biomolecular complexes in any real sense – the binding forces between the components seem to be rather weak and indiscriminate, and condensates typically contain proteins with some degree of disorder (intrinsically disordered proteins, IDPs), which tend to be promiscuous in their interactions. Debasis Saha and Biman Jana of the Indian Association for the Cultivation of Science in Kolkata have used MD simulations to investigate the factors that govern the interactions of model peptides, leading to dimerization, to try to get some handle on what is going on (J. Phys. Chem. B 127, 6656; 2023 - here). They consider the effect of charged residues such as arginine, as well as differing amounts of hydrophobicity in the chains, in altering the free energy surface for dimerization. For both positively and negatively charged peptides, the solvation water seems to play an important role, and is the dominant influence in the latter case. The upshot is that, if dimerization adequately reflects the condensate formation process, negatively charged peptides seem more likely to stay in the dilute phase.

Benjamin Schuler at Chicago and colleagues look at a similar issue: the interaction between the highly positively charged histone linker H1 and the highly negatively charged prothymosin α (ProTα) which acts as a “chaperone” that can help H1 disassociate from the histone (A. Chowdhury et al., PNAS 120, e2304036120; 2023 - here). Both are IDPs. They find that in this case there is a large entropic contribution to the binding coming from the release of counterions – a consideration that they expect to be most generally applicable to bio-polyelectrolyte interactions.  

Meanwhile, in a paper in bioRxiv [here], Saumyak Mukherjeee and Lars Schäfer at Bochum have looked at the thermodynamic driving forces that govern the formation of condensates from proteins with intrinsically disordered domains. Aggregation into the dense phase will involve changes in enthalpy and entropy due both to direct protein interactions and to changes in solvation. The authors conclude from MD simulations that in this case the most important factors are protein interaction enthalpy and the entropic effects of water release from the protein hydration shell into the bulk.

It seems likely that unravelling these factors governing IDP associations of various sorts will ultimately be valuable as we seek to make pharmaceutical interventions in molecular interactions of this kind that seem connected to disease (such as Alzheimer’s). Christine Lim at Cambridge and colleagues have developed a bioinformatics platform for identifying proteins involved in LLPS that seem to be potential therapeutic targets (C. M. Lim et al., PNAS 120, e2300215120; 2023 - here). They test it by looking at the in vitro phase behaviour of three targets that their scheme identifies. I don’t think it is too much to suggest that this points towards something of a new paradigm for therapeutics, in which the goal is not to develop some inhibitor that might compete with a protein’s normal ligand but rather to engineer collective and less selective interactions at a larger scale.

I’m intrigued by a paper in press in J. General Physiology (preprint here) by Alan Kay at the University of Iowa and Gerald Manning at Rutgers, arguing that what drives osmosis is still not fully understood and that the mechanism proposed by Peter Debye in 1923 is in fact the right one, despite being now largely forgotten. It is one thing to explain osmotic flow thermodynamically in terms of differences in chemical potential (the textbook account), but another to explain what actually drives the directional transport of water molecules. Manning and Kay say that diffusion alone is not able to account for the osmotic flux, which arises instead because of differential repulsive forces between the solute molecules and the two interfaces of the semipermeable membrane. This produces the equivalent of a hydrostatic pressure difference that drives the flux. I’m certainly not qualified to assess whether this revision of the textbook explanation is valid, but the history of the Debye model given in the paper is surely interesting in its own right.  

There is, admittedly, not much biology going on in the deep mantle of the Earth with temperatures of 1,000-2,000 K and pressures of up to 22 GPa. All the same, such conditions pose quite a test of molecular-dynamics water potential functions, which Roberto Car at Princeton, Giulia Galli at Chicago, and colleagues have put through their paces (C. Zhang et al., J. Phys. Chem B 127, 7011; 2023 - here). They set out to calculate the thermal conductivity of water in these conditions, using a potential found by fitting to density-functional theory using a deep-learning algorithm. The conductivity varies only slightly with temperature, decreasing from its ambient value, but is much more strongly (and positively) correlated with density. The heat transport of water at high P and T could have important implications for processes in the interiors of gas-giant planets.

It's back!

 

Back by popular demand” always seemed a self-congratulatory phrase, but in this case it really is the case that I’m reviving this blog because so many people have said they found it helpful. I’m touched by that, and also grateful to be given the motivation to continue pursuing this endlessly fascinating topic. So here we go.

 

One of the things that led me to let the blog languish was that there were so many other areas in which I have been striving to deepen my knowledge over the past several years. Happily, at least one of these remains relevant to the topic at hand: I have been delving deeper into the thickets of molecular and cell biology for my latest book, How Life Works, which is published in the autumn/fall of 2023. Among many other things, this looks at issues such as the roles of disordered proteins and of biomolecular condensates, in which issues of solvation are clearly important.

 

As if to underscore that, I am kicking off this post with a fascinating paper in Nature by Benjamin Schuler at Zurich and colleagues [N. Galvanetto et al., Nature 10.1038/s41586-023-06329-5; 2023], which looks at biomolecular dynamics within such a dense condensate. I probably don’t need now to say much about the importance of condensates in cell biology; as Schuler and colleagues say, these dense but loose associations of proteins and nucleic acids “play a key role in cellular processes operating at different scales, such as ribosome assembly, RNA splicing, stress response, mitosis and chromatin organization, and they are involved in a range of diseases” (and also, I’d add, in gene regulation). Their significance was confirmed by the fact that Tony Hyman and Cliff Brangwynne, who did much of the early work to bring them to wider attention, were awarded last year’s Breakthrough Prize in the life sciences. As Schuler et al. say, intrinsically disordered proteins seem often to play a key part in the formation of condensates, thanks to their rather promiscuous binding capacity.

 

The dense fluid of these phase-separated blobs can have a greatly enhanced local concentration of constituents relative to the bulk cytoplasm, and indeed this is a central feature of their function: they help to concentrate particular biomolecular species to enable repeated, low-affinity encounters of the kind that seem important for e.g. gene regulation or splicing. That density, however, results in a water viscosity that can be several orders of magnitude greater than it is in the bulk. Schuler and colleagues use single-molecule spectroscopy and MD simulations to examine the consequences of this for the dynamics of proteins. They study in vitro proxies for in vivo condensates, consisting of droplets (coacervates – I’m interested to see the return of that word from the days of the “prebiotic” speculations of Sidney Fox!) made from two highly and oppositely charged IDPs, both of which are known to influence chromatin condensation and transcriptional regulation.

 

The result is that, despite a 300-fold increase in water viscosity in the droplets and the formation of a dense network of indiscriminately associated proteins, leading to drastically slowed translational motion, the local conformational dynamics of the chains that influence residue-residue contacts remain fast, happening on pico- to nanosecond timescales. The conclusions are nicely summarized in this image:

 

 

As the authors say: “The behaviour we observe is an example of the subtle balance of

intermolecular interactions in biomolecular phase separation. On the one hand, the interactions must be strong enough for the formation of stable condensates; on the other hand, they need to be sufficiently weak to enable translational diffusion and liquid-like dynamics within the dense phase and molecular exchange across the phase boundary— processes that are essential for function, such as biochemical reactions occurring in condensates.” I have to say that this doesn’t entirely surprise me, as it seems to reflect in a more extreme way what seems to happen in cells more generally: the cytoplasm can look somewhat gel-like at mesoscales, and translational diffusion can be anomalous, while at the molecular scale dynamics are not so different from those in dilute solution. Of course, the usual caveats about in vitro studies – which have been stressed in particular for studies of condensates (see e.g here) – apply too. But this is a really nice study, which to my mind illustrates the possibility of a separation of dynamical timescales that makes life possible: the benefits of forming condensates do not need to come at the expense of compromising the local dynamics needed for biomolecular function.

 

Needless to say, I have from time to time seen some wonderful papers touching on water in biology come and go in the time between my last blog and now, and I fear these are lost now to being documented here. But there are a few recent ones that I still have to hand. There has been a particularly vigorous debate in the past year or two on the issue of the putative liquid-liquid phase transition in pure water. The evidence for this continues to accumulate, and looks now to be rather strong – although that clinching proof remains elusive. This paper [Science 359, 1127; 2018] by Sander Woutersen in Amsterdam – including the late and much missed Austen Angell – reported a direct sighting of such a transition in an aqueous solution of hydrazinium trifluoroacetate, which could be deeply supercooled to 140 K. And work by Yoshiharu Suzuki in Tsukuba adds to that suggestive evidence with a study [PNAS 119, e2113411119; 2022] of supercooled aqueous trehalose, which reports a direct signature of a first-order liquid-liquid transition. You can see some responses to the work here. Meanwhile, Nguyen Vinh and colleagues at Virginia Tech have performed terahertz measurements of water over a wide temperature range, down to the deeply supercooled state, which they say can be interpreted it terms of two liquid forms with different transition temperatures.  

 

I am (not so?) secretly delighted that my PhD supervisor Bob Evans at Bristol decided some time ago to delve into water phase behaviour, having warned me (wisely!) to steer clear of any liquid so anomalous. Here [J. Chem. Phys. 158, 034508; 2023] Bob, Mary Coe and Nigel Wilding suggest that the well known density depletion and enhanced fluctuations evident in water near hydrophobic surfaces be interpreted as the remnant of a critical surface (drying, or as some call it, dewetting) phase transition. In other words – and this seems to me quite significant – there is in a sense nothing unusual about water in this respect, and no need to invoke any specialness about its liquid-state structure. Rather, this is well-understood statistical physics at work. I notice that this was the topic of Mary’s 2021 PhD thesis: “Hydrophobicity across length scales: The role of surface criticality” – which I’d love to see.

 

I can’t touch on that topic without remembering that for my last post in January 2018, I seem not yet to have caught up with the news of the death in April 2017 of David Chandler, a giant in the field of liquid-state theory, as well as statistical mechanics more broadly. David’s work with John Weeks and Ka Lum on the hydrophobic interaction [JPCB 103, 4570; 1999] has of course been one of the most influential contributions to this topic since it was published in 1999. David’s work has certainly been a huge influence on my thinking in this field – and indeed that influence went right back to my work with Bob in the mid-1980s. David was always generous but rigorous, and phenomenally insightful, and his absence is deeply felt.

 

Francesco Paesani of UCSD has let me know about the new water potential he and his colleagues have developed from first-principles quantum simulations, which is able to capture the phase diagram very realistically [Bore & Paesani, Nat. Commun. 14, 3349; 2023] – see also the improvements in Zhu et al., J. Chem. Theory Comput. 19, 3551-3566; 2023]. Francesco has already been applying it to a variety of real-world problems, including the hydration of a simple model of the protein backbone (N-methylacetamide) [Zhou et al., J. Chem. Theory Comput. 19, 4308; 2023].

 

I just noticed this interesting article [A. Katsnelson, ACS Cent. Sci. 10.1021/acscentsci.3c00803; 2023] on “artificial saliva” and synthetic mucins. I knew nothing about such work, which sound like rich ground for exploring hydration issues.

 

I can’t end this first post without mentioning the ongoing story of “life (or not) in the clouds of Venus”. This intriguing idea was first put forward by Sara Seager and her colleagues following the apparent identification [J. S. Greaves et al., Nat. Astron. 5, 655; 2021] of phosphine – for which no abiotic natural source is known – in the Venusian atmosphere. Much of the immediate debate (aside from that about how secure the signature of phosphine really was, a discussion that is still ongoing) centred on the extreme acidity of the cloud droplets. But I was more concerned about how low the water activity in the droplets must be – we know that no life on Earth has been seen for water activities below 0.585, a fact that I am sure is related to the minimal hydration requirements for functional biomolecules. When I mentioned this to microbiologist John Hallsworth at Queens in Belfast, he convened a team of experts who made calculations to estimate the water activity in the Venusian clouds and a variety of other planetary environments, revealing that on Venus this is indeed orders of magnitude too low to permit terrestrial-type life given what we currently know about it [J. E. Hallsworth et al., Nat. Astron. 5, 665; 2021]. Sara and her coworkers have now suggested [W. Bains et al., Astrobiol. 10.1089/ast.2022.0113; 2023] possible ways around this problem, which in the end amount to having to posit life unlike that on Earth – a possibility we recognized in our paper. To my mind, this could usefully sharpen the discussion about what “habitable” can and should mean in astrobiology, as I explore here. I welcome anything that prompts deeper, quantitative thinking about the conditions needed to support something we might reasonably call life in extraterrestrial environments. But I would argue that such discussions need to go beyond “well, X can also act as a hydrogen-bonding solvent”, and should take into consideration just how subtle water’s roles are in terrestrial biochemistry, and how much the “specialness” of water might matter in this regard. Given the richness of emerging knowledge about extraterrestrial environments, and the prospects of much more to come through planned missions (e.g. to Enceladus and Titan) and JWST observations, I’d love to see this issue engaged with head-on by e.g. ESA or NASA.   

 

Well, there we are: I’m open for business again. So do feel free to send things my way for inclusion; I’d be delighted to hear about them.