Dynamic Structural Changes and Thermodynamics in Phase Separation Processes of an Intrinsically Disordered-Ordered Protein Model.
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ABSTRACT:
SUBMITTER: Ludeke S
PROVIDER: S-EPMC9299898 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The phase separation behavior of intrinsically disordered proteins (IDPs) is thought of as analogous to that of polymers that undergo equilibrium lower or upper critical solution temperature (LCST and UCST, respectively) phase transition. This view, however, ignores possible nonequilibrium properties of protein assemblies. Here, by studying IDP polymers (IDPPs) composed of repeat motifs that encode LCST or UCST phase behavior, we discovered that IDPs can access a wide spectrum of nonequilibrium, hysteretic phase behaviors. Experimentally and through simulations, we show that hysteresis in IDPPs is tunable and that it emerges through increasingly stable interchain interactions in the insoluble phase. To explore the utility of hysteretic IDPPs, we engineer self-assembling nanostructures with tunable stability. These findings shine light on the rich phase separation behavior of IDPs and illustrate hysteresis as a design parameter to program nonequilibrium phase behavior in self-assembling materials.
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Project description:RXRβ is one of three subtypes of human retinoid X receptor (RXR), a transcription factor that belongs to the nuclear receptor superfamily. Its expression can be detected in almost all tissues. In contrast to other subtypes - RXRα and RXRγ - RXRβ has the longest and unique N-terminal sequence called the AB region, which harbors a ligand-independent activation function. In contrast to the functional properties of this sequence, the molecular properties of the AB region of human RXRβ (AB_hRXRB) have not yet been characterized. Here, we present a systematic biochemical and biophysical analysis of recombinant AB_hRXRB, along with in silico examinations, which demonstrate that AB_hRXRB exhibits properties of a coil-like intrinsically disordered region. AB_hRXRB possesses a flexible structure that is able to adopt a more ordered conformation under the influence of different environmental factors. Interestingly, AB_hRXRB promotes the formation of liquid-liquid phase separation (LLPS), a phenomenon previously observed for the AB region of another human subtype of RXR - RXRγ (AB_hRXRG). Although both AB regions seem to be similar in terms of their ability to induce phase separation, they clearly differ in the sensitivity to factors driving and regulating LLPS. This distinct LLPS response to environmental factors driven by the unique amino acid compositions of AB_hRXRB and AB_hRXRG can be significant for the specific modulation of the transcriptional activation of target genes by different subtypes of RXR. Video Abstract.
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Project description:Protein phase separation is thought to be a primary driving force for the formation of membrane-less organelles, which control a wide range of biological functions from stress response to ribosome biogenesis. Among phase-separating (PS) proteins, many have intrinsically disordered regions (IDRs) that are needed for phase separation to occur. Accurate identification of IDRs that drive phase separation is important for testing the underlying mechanisms of phase separation, identifying biological processes that rely on phase separation, and designing sequences that modulate phase separation. To identify IDRs that drive phase separation, we first curated datasets of folded, ID, and PS ID sequences. We then used these sequence sets to examine how broadly existing amino acid property scales can be used to distinguish between the three classes of protein regions. We found that there are robust property differences between the classes and, consequently, that numerous combinations of amino acid property scales can be used to make robust predictions of protein phase separation. This result indicates that multiple, redundant mechanisms contribute to the formation of phase-separated droplets from IDRs. The top-performing scales were used to further optimize our previously developed predictor of PS IDRs, ParSe. We then modified ParSe to account for interactions between amino acids and obtained reasonable predictive power for mutations that have been designed to test the role of amino acid interactions in driving protein phase separation. Collectively, our findings provide further insight into the classification of IDRs and the elements involved in protein phase separation.
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Project description:The abundance of intrinsic disorder in the protein realm and its role in a variety of physiological and pathological cellular events have strengthened the interest of the scientific community in understanding the structural and dynamical properties of intrinsically disordered proteins (IDPs) and regions (IDRs). Attempts at rationalizing the general principles underlying both conformational properties and transitions of IDPs/IDRs must consider the abundance of charged residues (Asp, Glu, Lys, and Arg) that typifies these proteins, rendering them assimilable to polyampholytes or polyelectrolytes. Their conformation strongly depends on both the charge density and distribution along the sequence (i.e., charge decoration) as highlighted by recent experimental and theoretical studies that have introduced novel descriptors. Published experimental data are revisited herein in the frame of this formalism, in a new and possibly unitary perspective. The physicochemical properties most directly affected by charge density and distribution are compaction and solubility, which can be described in a relatively simplified way by tools of polymer physics. Dissecting factors controlling such properties could contribute to better understanding complex biological phenomena, such as fibrillation and phase separation. Furthermore, this knowledge is expected to have enormous practical implications for the design, synthesis, and exploitation of bio-derived materials and the control of natural biological processes.
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Project description: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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