Significance: Liquid–liquid phase separation of proteins results in biomolecular condensates, which contribute to the organization of cellular matter into membraneless organelles. It is still unclear, however, whether these condensates represent a common state of proteins. Here, based on biophysical principles driving phase separation, we report a proteome-wide ranking of proteins according to their propensity to condensate into a droplet state. We analyze two mechanisms for droplet formation—driver proteins can spontaneously phase separate, while client proteins require additional components. We conclude that the droplet state, as the native and amyloid states, is a fundamental state of proteins, with most proteins expected to be capable of undergoing liquid–liquid phase separation via either of these two mechanisms.

Specific molecular recognition is assumed to require a well‐defined set of contacts and devoid of conformational and interaction ambiguities. Growing experimental evidence demonstrates however, that structural multiplicity or dynamic disorder can be retained in protein complexes, termed as fuzziness. Fuzzy regions establish alternative contacts between specific partners usually via transient interactions. Nature often tailors the dynamic properties of these segments via post‐translational modifications or alternative splicing to fine‐tune affinity. Most experimentally characterized fuzzy complexes are involved in regulation of gene‐expression, signal transduction and cell‐cycle regulation. Fuzziness is also characteristic to viral protein complexes, cytoskeleton structure, and surprisingly in a few metabolic enzymes. A plausible role of fuzzy complexes in increasing half‐life of intrinsically disordered proteins is also discussed.

Many proteins perform their functions within membraneless organelles, where they form a liquid-like condensed state, also known as droplet state. The FuzDrop method predicts the probability of spontaneous liquid–liquid phase separation of proteins and provides a sequence-based score to identify the regions that promote this process. Furthermore, the FuzDrop method estimates the propensity of conversion of proteins to the amyloid state, and identifies aggregation hot-spots, which can drive the irreversible maturation of the liquid-like droplet state. These predictions can also identify mutations that can induce formation of amyloid aggregates, including those implicated in human diseases. To facilitate the interpretation of the predictions, the droplet-promoting and aggregation-promoting regions can be visualized on protein structures generated by AlphaFold. The FuzDrop server (https://fuzdrop.bio.unipd.it) thus offers insights into the complex behavior of proteins in their condensed states and facilitates the understanding of the functional relationships of proteins.

Are all protein interactions fully optimized? Do suboptimal interactions compromise specificity? What is the functional impact of frustration? Why does evolution not optimize some contacts? Proteins and their complexes are best described as ensembles of states populating an energy landscape. These ensembles vary in breadth from narrow ensembles clustered around a single average X-ray structure to broader ensembles encompassing a few different functional “taxonomic” states on to near continua of rapidly interconverting conformations, which are called “fuzzy” or even “intrinsically disordered”. Here we aim to provide a comprehensive framework for confronting the structural and dynamical continuum of protein assemblies by combining the concepts of energetic frustration and interaction fuzziness. The diversity of the protein structural ensemble arises from the frustrated conflicts between the interactions that create the energy landscape. When frustration is minimal after folding, it results in a narrow ensemble, but residual frustrated interactions result in fuzzy ensembles, and this fuzziness allows a versatile repertoire of biological interactions. Here we discuss how fuzziness and frustration play off each other as proteins fold and assemble, viewing their significance from energetic, functional, and evolutionary perspectives. We demonstrate, in particular, that the common physical origin of both concepts is related to the ruggedness of the energy landscapes, intramolecular in the case of frustration and intermolecular in the case of fuzziness. Within this framework, we show that alternative sets of suboptimal contacts may encode specificity without achieving a single structural optimum. Thus, we demonstrate that structured complexes may not be optimized, and energetic frustration is realized via different sets of contacts leading to multiplicity of specific complexes. Furthermore, we propose that these suboptimal, frustrated, or fuzzy interactions are under evolutionary selection and expand the biological repertoire by providing a multiplicity of biological activities. In accord, we show that non-native interactions in folding or interaction landscapes can cooperate to generate diverse functional states, which are essential to facilitate adaptation to different cellular conditions. Thus, we propose that not fully optimized structures may actually be beneficial for biological activities of proteins via an alternative set of suboptimal interactions. The importance of such variability has not been recognized across different areas of biology. This account provides a modern view on folding, function, and assembly across the protein universe. The physical framework presented here is applicable to the structure and dynamics continuum of proteins and opens up new perspectives for drug design involving not fully structured, highly dynamic protein assemblies.