What is it about?

A key feature of living cells is the ability to carry out biological reactions at defined locations and at the right time. One of the traditionally well-known and established mechanisms to achieve this spatial (in space) and temporal (in time) regulation of biochemical reactions is by confining the relevant proteins/nucleic acids which carry out these reactions within an enclosed space that is defined by a membrane. These structures are known as organelles, and among the textbook examples of organelles include the Golgi apparatus, the endoplasmic reticulum and the mitochondria. These membrane-enclosed organelles are largely permanent and persist over the lifespan of a cell. However, more recent developments in cell biology have helped shed light on the existence of a second group of organelles which are not physically defined by a membrane and hence referred to as membrane-less organelles. Unlike the membrane-enclosed organelles, membrane-less organelles can assemble at certain stages of a cell’s lifetime or in response to environmental cues such as heat or chemical stress. Once these cues go away these membrane-less organelles disassemble, making dynamics a key aspect of their function.  In this work, we explore how a key molecular modification to proteins by the introduction of negative charges-- phosphorylation -- can regulate the dynamics and reversibility of these assemblies using molecular simulations and bioinformatics studies involving a known membrane-less organelle protein fused in-sarcoma (FUS). Our simulations and bioinformatics data show that the extent and pattern of modifications along a protein sequence can influence the dynamics and outcome of FUS assemblies.

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Why is it important?

Biomolecular assemblies such as membrane-less organelles are essentially dense pockets of proteins and, often, nucleic acids (DNA, RNA) which assemble due to favorable adhesive interactions between these biomolecules. To drive the formation of these structures, protein sequences (a string of building blocks known as amino acids) have to evolve in such a manner as to ensure sufficient adhesive interactions hold the assembly in place. If the interactions are too weak, the proteins would prefer to remain as individual units in the cytoplasm and, therefore, unable to perform their native function as a collective. On the other hand, if the interactions are too strong or “sticky”, the ensuing structures would be incapable of disassembly in the absence of external stresses, making them irreversible. Such irreversible, solid-like assemblies are often detrimental to the cell, hampering their healthy function. Indeed, irreversible protein deposits are associated with neurodegenerative diseases such as Alzheimer’s’ Parkinson's and ALS. How, then, do cells manage to achieve the assembly of membrane-less organelles while safeguarding against irreversible protein deposits? Our bioinformatics analysis shows that mammalian FUS protein sequences contain an unusually high content of solidification-prone amino-acid stretches known as amyloidogenic sequences. The fraction of amyloid-prone stretches in mammalian sequences is significantly higher than if they were to occur randomly with no evolutionary bias. Interestingly, consistent with the higher amyloidogenic content, mammalian sequences are also significantly enriched in sequences that are prone to phosphorylation. Phosphorylation is a protein modification that increases the net negative charge of the protein resulting in stronger repulsive interactions between different protein molecules (with the same sequence). Crucially, we also observed a strong correlation between the location of the amyloid-prone sequence and the phosphorylation site placement. Using molecular simulations, we also established that a variation in the extent, and location of phosphorylation sites can also significantly alter the fate of the FUS assemblies and tune their dynamics differentially and also result in disassembly in some cases. These results are crucial from an evolutionary biology standpoint and to understand the native evolutionary safeguards against aberrant protein assemblies leading to degenerative disorders. Understanding these safeguarding mechanisms and evolutionary checkpoints could also be a key element while devising intervention strategies against degenerative diseases.


The ability to tune and regulate biological interactions is vital for cells to be able to respond effectively to environmental cues. Over billions of years of evolution, cells have evolved several different mechanisms to achieve effective response mechanisms. Post-translational modifications such as phosphorylation can offer cells a dynamic handle to tune the physicochemical properties of protein sequences, offering a molecular mechanism to respond to external stimuli. Our results are yet another example of how living systems operate at the edge of chaos and are susceptible to subtle molecular perturbations.

Dr Srivastav Ranganathan
Harvard University

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This page is a summary of: Phosphorylation sites are evolutionary checkpoints against liquid–solid transition in protein condensates, Proceedings of the National Academy of Sciences, May 2023, Proceedings of the National Academy of Sciences, DOI: 10.1073/pnas.2215828120.
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