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1. Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior

   https://doi.org/10.1073/pnas.2000223117  

   Schuster, Benjamin S.; Dignon, Gregory L.; Tang, Wai Shing; Kelley, Fleurie M.; Ranganath, Aishwarya Kanchi; Jahnke, Craig N.; Simpkins, Alison G.; Regy, Roshan Mammen; Hammer, Daniel A.; Good, Matthew C.; et al (May 2020, Proceedings of the National Academy of Sciences)

   null (Ed.)

   Phase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bioinspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. We designed sequences with significantly increased phase separation propensity by shuffling the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation. This result indicates the natural sequence is under negative selection to moderate this mode of interaction. We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that despite these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance our fundamental understanding of key biophysical principles and sequence features important to phase separation. 

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2. Intramolecular interactions between folded and disordered regions shape ubiquilin structure and function

   https://doi.org/10.64898/2026.03.13.711692  

   Niblo, Jessica K; Acharya, Nirbhik; Watkins, Maxwell B; Castañeda, Carlos A; Sukenik, Shahar (March 2026, bioRxiv)

   Abstract Multidomain proteins consist of folded domains connected by intrinsically disordered regions. The flexibility afforded by the disordered regions coupled to the structure and surface chemistry of folded regions allows for unique structural and functional features in these proteins. Yet how intramolecular interactions between disordered regions and folded domains affect multidomain protein structure and function remain poorly understood. Here we use a range of biophysical and computational approaches to measure the intramolecular interactions between the folded domains and disordered regions of ubiquilins (UBQLNs) - essential components of protein quality control that shuttle poly-ubiquitinated client proteins to proteasomal degradation or autophagy. Starting with the yeast UBQLN homolog Dsk2, we find that interactions between two folded domains located at the opposite ends of UBQLN bring about a closed conformation. The prevalence of this closed conformation, however, is modulated by intramolecular interactions involving the disordered regions and folded STI1 domain at the center of the protein. Simulations and analysis of UBQLN homologs across multiple eukaryotic lineages reveals that these disordered:folded domain interactions exist in some UBQLN homologs but are absent in others, indicating possible fundamental differences in function among proteins with the same multidomain architecture. 

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3. Charge Distribution Patterns of IA ₃ Impact Conformational Expansion and Hydration Diffusivity of the Disordered Ensemble

   https://doi.org/10.1021/acs.jpcb.3c06170  

   Dunleavy, Katie M.; Li, Tianyan; Milshteyn, Eugene; Jaufer, Afnan M.; Walker, Shamon A.; Fanucci, Gail E. (November 2023, The Journal of Physical Chemistry B)

   Dr. Sudipta Maiti (Ed.)

   IA3 is a 68 amino acid natural peptide/protein inhibitor of yeast aspartic proteinase A (YPRA) that is intrinsically dis-ordered in solution with induced N-terminal helicity when in the protein complex with YPRA. Based upon the intrinsical-ly disordered proteins (IDPs) parameters of fractional net charge (FNC), of net charge density per residue (NCPR) and of charge patterning (), the two domains of IA3 are defined to occupy different domains within conformationally based subclasses of IDPs; thus, making IA3 a bimodal-domain IDP. Site-directed spin-labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and low-field Overhauser dynamic nuclear polarization (ODNP) spectroscopy results show that these two domains possess different degrees of compaction and hydration diffusivity behavior. This work suggests that SDSL EPR line shapes – analyzed in terms of their local tumbling volume (VL) – provide insight into the compaction of the unstructured IDP ensemble in solution and that protein sequence and net charge distribution pat-terns within a conformational subclass can impact bound water hydration dynamics; thus, possibly offering an alter-native thermodynamic property that can encode conforma-tional binding and behavior of IDPs and liquid-liquid phase separations. 

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4. Sizes, conformational fluctuations, and SAXS profiles for intrinsically disordered proteins

   https://doi.org/10.1002/pro.70067  

   Mugnai, Mauro L; Chakraborty, Debayan; Nguyen, Hung T; Maksudov, Farkhad; Kumar, Abhinaw; Zeno, Wade; Stachowiak, Jeanne C; Straub, John E; Thirumalai, D (April 2025, Protein Science)

   Abstract The preponderance of intrinsically disordered proteins (IDPs) in the eukaryotic proteome, and their ability to interact with each other, and with folded proteins, RNA, and DNA for functional purposes, have made it important to quantitatively characterize their biophysical properties. Toward this end, we developed the transferable self‐organized polymer (SOP‐IDP) model to calculate the properties of several IDPs. The values of the radius of gyration () obtained from SOP‐IDP simulations are in excellent agreement (correlation coefficient of 0.96) with those estimated from SAXS experiments. For AP180 and Epsin, the predicted values of the hydrodynamic radii () are in nearly quantitative agreement with those from fluorescence correlation spectroscopy (FCS) experiments. Strikingly, the calculated SAXS profiles for 36 IDPs are also nearly superimposable on the experimental profiles. The dependence of and the mean end‐to‐end distance () on chain length, , follows Flory's scaling law, ( and ), suggesting that globally IDPs behave as synthetic polymers in a good solvent. This finding depends on the solvent quality, which can be altered by changing variables such as pH and salt concentration. The values of and are 0.20 and 0.48 nm, respectively. Surprisingly, finite size corrections to scaling, expected on theoretical grounds, are negligible for and . In contrast, only by accounting for the finite sizes of the IDPs, the dependence of experimentally measurable on can be quantitatively explained using . Although Flory scaling law captures the estimates for , , and accurately, the spread of the simulated data around the theoretical curve is suggestive of of sequence‐specific features that emerge through a fine‐grained analysis of the conformational ensembles using hierarchical clustering. Typically, the ensemble of conformations partitions into three distinct clusters, having different equilibrium populations and structural properties. Without any further readjustments to the parameters of the SOP‐IDP model, we also obtained nearly quantitative agreement with paramagnetic relaxation enhancement (PRE) measurements forα‐synuclein. The transferable SOP‐IDP model sets the stage for several applications, including the study of phase separation in IDPs and interactions with nucleic acids. 

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5. Role of Charge Patterning and Hydrophobicity in Peptide-Based Complex Coacervates

   https://doi.org/10.1021/acs.biomac.5c00773  

   Sathyavageeswaran, Arvind; Pandey, Pankaj Kumar; Holmlund, Nickolas; Kaushik, Priyanka; McIntosh, Shannon; Mokdad, Raneem; Perry, Sarah L (October 2025, Biomacromolecules)

   Complex coacervation has emerged as a powerful model for studying the self-assembly of intrinsically disordered proteins (IDPs) in biological condensates in cells. We characterized the phase behavior and rheology of coacervates formed from peptides with regular repeating sequences to examine the effects of charge patterning and hydrophobicity on coacervate stability and material properties. Our results show that increasing the size of charged blocks enhances salt resistance via electrostatic cooperativity, while incorporating small hydrophobic segments further stabilizes coacervates and increases viscosity through hydrophobic clustering. Interestingly, our results involving alanine as the neutral residue suggest that structural conformations present for peptides with shorter block sizes may contribute to increased viscosity within the coacervate phase. Overall, these findings underscore the potential of sequence-controlled materials as versatile platforms for engineering self-assembling materials with tunable mechanical and phase properties. 

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