Isolation of nucleic acids using liquid-liquid phase separation of pH-sensitive elastin-like polypeptides.

Extraction of nucleic acids (NAs) is critical for many methods in molecular biology and bioanalytical chemistry. NA extraction has been extensively studied and optimized for a wide range of applications and its importance to society has significantly increased. The COVID-19 pandemic highlighted the importance of early and efficient NA testing, for which NA extraction is a critical analytical step prior to the detection by methods like polymerase chain reaction. This study explores simple, new approaches to extraction using engineered smart nanomaterials, namely NA-binding, intrinsically disordered proteins (IDPs), that undergo triggered liquid-liquid phase separation (LLPS). Two types of NA-binding IDPs are studied, both based on genetically engineered elastin-like polypeptides (ELPs), model IDPs that exhibit a lower critical solution temperature in water and can be designed to exhibit LLPS at desired temperatures in a variety of biological solutions. We show that ELP fusion proteins with natural NA-binding domains can be used to extract DNA and RNA from physiologically relevant solutions. We further show that LLPS of pH responsive ELPs that incorporate histidine in their sequences can be used for both binding, extraction and release of NAs from biological solutions, and can be used to detect SARS-CoV-2 RNA in samples from COVID-positive patients.

Unveiling the Binding between the Armadillo-Repeat Domain of Plakophilin 1 and the Intrinsically Disordered Transcriptional Repressor RYBP.

Plakophilin 1 (PKP1), a member of the p120ctn subfamily of the armadillo (ARM)-repeat-containing proteins, is an important structural component of cell-cell adhesion scaffolds although it can also be ubiquitously found in the cytoplasm and the nucleus. RYBP (RING 1A and YY1 binding protein) is a multifunctional intrinsically disordered protein (IDP) best described as a transcriptional regulator. Both proteins are involved in the development and metastasis of several types of tumors. We studied the binding of the armadillo domain of PKP1 (ARM-PKP1) with RYBP by using in cellulo methods, namely immunofluorescence (IF) and proximity ligation assay (PLA), and in vitro biophysical techniques, namely fluorescence, far-ultraviolet (far-UV) circular dichroism (CD), and isothermal titration calorimetry (ITC). We also characterized the binding of the two proteins by using in silico experiments. Our results showed that there was binding in tumor and non-tumoral cell lines. Binding in vitro between the two proteins was also monitored and found to occur with a dissociation constant in the low micromolar range (~10 µM). Finally, in silico experiments provided additional information on the possible structure of the binding complex, especially on the binding ARM-PKP1 hot-spot. Our findings suggest that RYBP might be a rescuer of the high expression of PKP1 in tumors, where it could decrease the epithelial-mesenchymal transition in some cancer cells.

Copper(II) coordination to the intrinsically disordered region of SARS-CoV-2 Nsp1.

The intrinsically disordered C-terminal peptide region of severe acute respiratory syndrome coronavirus 2 nonstructural protein-1 (Nsp1-CT) inhibits host protein synthesis by blocking messenger RNA (mRNA) access to the 40S ribosome entrance tunnel. Aqueous copper(II) ions bind to the disordered peptide with micromolar affinity, creating a possible strategy to restore protein synthesis during host infection. Electron paramagnetic resonance (EPR) and tryptophan fluorescence measurements on a 10-residue model of the disordered protein region (Nsp1-CT10), combined with advanced quantum mechanics calculations, suggest that the peptide binds to copper(II) as a multidentate ligand. Two optimized computational models of the copper(II)-peptide complexes were derived: One corresponding to pH 6.5 and the other describing the complex at pH 7.5 to 8.5. Simulated EPR spectra based on the calculated model structures are in good agreement with experimental spectra.

Structural studies of intrinsically disordered MLL-fusion protein AF9 in complex with peptidomimetic inhibitors.

AF9 (MLLT3) and its paralog ENL(MLLT1) are members of the YEATS family of proteins with important role in transcriptional and epigenetic regulatory complexes. These proteins are two common MLL fusion partners in MLL-rearranged leukemias. The oncofusion proteins MLL-AF9/ENL recruit multiple binding partners, including the histone methyltransferase DOT1L, leading to aberrant transcriptional activation and enhancing the expression of a characteristic set of genes that drive leukemogenesis. The interaction between AF9 and DOT1L is mediated by an intrinsically disordered C-terminal ANC1 homology domain (AHD) in AF9, which undergoes folding upon binding of DOT1L and other partner proteins. We have recently reported peptidomimetics that disrupt the recruitment of DOT1L by AF9 and ENL, providing a proof-of-concept for targeting AHD and assessing its druggability. Intrinsically disordered proteins, such as AF9 AHD, are difficult to study and characterize experimentally on a structural level. In this study, we present a successful protein engineering strategy to facilitate structural investigation of the intrinsically disordered AF9 AHD domain in complex with peptidomimetic inhibitors by using maltose binding protein (MBP) as a crystallization chaperone connected with linkers of varying flexibility and length. The strategic incorporation of disulfide bonds provided diffraction-quality crystals of the two disulfide-bridged MBP-AF9 AHD fusion proteins in complex with the peptidomimetics. These successfully determined first series of 2.1-2.6 Å crystal complex structures provide high-resolution insights into the interactions between AHD and its inhibitors, shedding light on the role of AHD in recruiting various binding partner proteins. We show that the overall complex structures closely resemble the reported NMR structure of AF9 AHD/DOT1L with notable difference in the conformation of the ß-hairpin region, stabilized through conserved hydrogen bonds network. These first series of AF9 AHD/peptidomimetics complex structures are providing insights of the protein-inhibitor interactions and will facilitate further development of novel inhibitors targeting the AF9/ENL AHD domain.

Hydrodynamic Radii of Intrinsically Disordered Proteins: Fast Prediction by Minimum Dissipation Approximation and Experimental Validation.

The diffusion coefficients of globular and fully unfolded proteins can be predicted with high accuracy solely from their mass or chain length. However, this approach fails for intrinsically disordered proteins (IDPs) containing structural domains. We propose a rapid predictive methodology for estimating the diffusion coefficients of IDPs. The methodology uses accelerated conformational sampling based on self-avoiding random walks and includes hydrodynamic interactions between coarse-grained protein subunits, modeled using the generalized Rotne-Prager-Yamakawa approximation. To estimate the hydrodynamic radius, we rely on the minimum dissipation approximation recently introduced by Cichocki et al. Using a large set of experimentally measured hydrodynamic radii of IDPs over a wide range of chain lengths and domain contributions, we demonstrate that our predictions are more accurate than the Kirkwood approximation and phenomenological approaches. Our technique may prove to be valuable in predicting the hydrodynamic properties of both fully unstructured and multidomain disordered proteins.

Linear and Nonlinear Dielectric Response of Intrinsically Disordered Proteins.

Linear and nonlinear dielectric responses of solutions of intrinsically disordered proteins (IDPs) were analyzed by combining molecular dynamics simulations with formal theories. A large increment of the linear dielectric function over that of the solvent is found and related to large dipole moments of IDPs. The nonlinear dielectric effect (NDE) of the IDP far exceeds that of the bulk electrolyte, offering a route to interrogate protein conformational and rotational statistics and dynamics. Conformational flexibility of the IDP makes the dipole moment statistics consistent with the gamma/log-normal distributions and contributes to the NDE through the dipole moment's non-Gaussian parameter. The intrinsic non-Gaussian parameter of the dipole moment combines with the protein osmotic compressibility in the nonlinear dielectric susceptibility when dipolar correlations are screened by the electrolyte. The NDE is dominated by dipolar correlations when electrolyte screening is reduced.

Synthetic intrinsically disordered protein fusion tags that enhance protein solubility.

We report the de novo design of small (<20 kDa) and highly soluble synthetic intrinsically disordered proteins (SynIDPs) that confer solubility to a fusion partner with minimal effect on the activity of the fused protein. To identify highly soluble SynIDPs, we create a pooled gene-library utilizing a one-pot gene synthesis technology to create a large library of repetitive genes that encode SynIDPs. We identify three small (<20 kDa) and highly soluble SynIDPs from this gene library that lack secondary structure and have high solvation. Recombinant fusion of these SynIDPs to three known inclusion body forming proteins rescue their soluble expression and do not impede the activity of the fusion partner, thereby eliminating the need for removal of the SynIDP tag. These findings highlight the utility of SynIDPs as solubility tags, as they promote the soluble expression of proteins in E. coli and are small, unstructured proteins that minimally interfere with the biological activity of the fused protein.

Understanding the Energy Landscape of Intrinsically Disordered Protein Ensembles.

A substantial portion of various organisms' proteomes comprises intrinsically disordered proteins (IDPs) that lack a defined three-dimensional structure. These IDPs exhibit a diverse array of conformations, displaying remarkable spatiotemporal heterogeneity and exceptional conformational flexibility. Characterizing the structure or structural ensemble of IDPs presents significant conceptual and methodological challenges owing to the absence of a well-defined native structure. While databases such as the Protein Ensemble Database (PED) provide IDP ensembles obtained through a combination of experimental data and molecular modeling, the absence of reaction coordinates poses challenges in comprehensively understanding pertinent aspects of the system. In this study, we leverage the energy landscape visualization method (JCTC, 6482, 2019) to scrutinize four IDP ensembles sourced from PED. ELViM, a methodology that circumvents the need for a priori reaction coordinates, aids in analyzing the ensembles. The specific IDP ensembles investigated are as follows: two fragments of nucleoporin (NUL: 884-993 and NUS: 1313-1390), yeast sic 1 N-terminal (1-90), and the N-terminal SH3 domain of Drk (1-59). Utilizing ELViM enables the comprehensive validation of ensembles, facilitating the detection of potential inconsistencies in the sampling process. Additionally, it allows for identifying and characterizing the most prevalent conformations within an ensemble. Moreover, ELViM facilitates the comparative analysis of ensembles obtained under diverse conditions, thereby providing a powerful tool for investigating the functional mechanisms of IDPs.

The Streptococcus phage protein paratox is an intrinsically disordered protein.

The bacteriophage protein paratox (Prx) blocks quorum sensing in its streptococcal host by directly binding the signal receptor and transcription factor ComR. This reduces the ability of Streptococcus to uptake environmental DNA and protects phage DNA from damage by recombination. Past work characterizing the Prx:ComR molecular interaction revealed that paratox adopts a well-ordered globular fold when bound to ComR. However, solution-state biophysical measurements suggested that Prx may be conformationally dynamic. To address this discrepancy, we investigated the stability and dynamic properties of Prx in solution using circular dichroism, nuclear magnetic resonance, and several fluorescence-based protein folding assays. Our work shows that under dilute buffer conditions Prx is intrinsically disordered. We also show that the addition of kosmotropic salts or protein stabilizing osmolytes induces Prx folding. However, the solute stabilized fold is different from the conformation Prx adopts when it is bound to ComR. Furthermore, we have characterized Prx folding thermodynamics and folding kinetics through steady-state fluorescence and stopped flow kinetic measurements. Our results show that Prx is a highly dynamic protein in dilute solution, folding and refolding within the 10 ms timescale. Overall, our results demonstrate that the streptococcal phage protein Prx is an intrinsically disordered protein in a two-state equilibrium with a solute-stabilized folded form. Furthermore, the solute-stabilized fold is likely the predominant form of Prx in a solute-crowded bacterial cell. Finally, our work suggests that Prx binds and inhibits ComR, and thus quorum sensing in Streptococcus, by a combination of conformational selection and induced-fit binding mechanisms.

Tardigrade secretory proteins protect biological structures from desiccation.

Tardigrades, microscopic animals that survive a broad range of environmental stresses, express a unique set of proteins termed tardigrade-specific intrinsically disordered proteins (TDPs). TDPs are often expressed at high levels in tardigrades upon desiccation, and appear to mediate stress adaptation. Here, we focus on the proteins belonging to the secreted family of tardigrade proteins termed secretory-abundant heat soluble ("SAHS") proteins, and investigate their ability to protect diverse biological structures. Recombinantly expressed SAHS proteins prevent desiccated liposomes from fusion, and enhance desiccation tolerance of E. coli and Rhizobium tropici upon extracellular application. Molecular dynamics simulation and comparative structural analysis suggest a model by which SAHS proteins may undergo a structural transition upon desiccation, in which removal of water and solutes from a large internal cavity in SAHS proteins destabilizes the beta-sheet structure. These results highlight the potential application of SAHS proteins as stabilizing molecules for preservation of cells.

AlphaFold2 modeling and molecular dynamics simulations of an intrinsically disordered protein.

We use AlphaFold2 (AF2) to model the monomer and dimer structures of an intrinsically disordered protein (IDP), Nvjp-1, assisted by molecular dynamics (MD) simulations. We observe relatively rigid dimeric structures of Nvjp-1 when compared with the monomer structures. We suggest that protein conformations from multiple AF2 models and those from MD trajectories exhibit a coherent trend: the conformations of an IDP are deviated from each other and the conformations of a well-folded protein are consistent with each other. We use a residue-residue interaction network (RIN) derived from the contact map which show that the residue-residue interactions in Nvjp-1 are mainly transient; however, those in a well-folded protein are mainly persistent. Despite the variation in 3D shapes, we show that the AF2 models of both disordered and ordered proteins exhibit highly consistent profiles of the pLDDT (predicted local distance difference test) scores. These results indicate a potential protocol to justify the IDPs based on multiple AF2 models and MD simulations.

Adenosine Triphosphate: The Primordial Molecule That Controls Protein Homeostasis and Shapes the Genome-Proteome Interface.

Adenosine triphosphate (ATP) acts as the universal energy currency that drives various biological processes, while nucleic acids function to store and transmit genetic information for all living organisms. Liquid-liquid phase separation (LLPS) represents the common principle for the formation of membrane-less organelles (MLOs) composed of proteins rich in intrinsically disordered regions (IDRs) and nucleic acids. Currently, while IDRs are well recognized to facilitate LLPS through dynamic and multivalent interactions, the precise mechanisms by which ATP and nucleic acids affect LLPS still remain elusive. This review summarizes recent NMR results on the LLPS of human FUS, TDP-43, and the viral nucleocapsid (N) protein of SARS-CoV-2, as modulated by ATP and nucleic acids, revealing the following: (1) ATP binds to folded domains overlapping with nucleic-acid-binding interfaces; (2) ATP and nucleic acids interplay to biphasically modulate LLPS by competitively binding to overlapping pockets of folded domains and Arg/Lys within IDRs; (3) ATP energy-independently induces protein folding with the highest efficiency known so far. As ATP likely emerged in the prebiotic monomeric world, while LLPS represents a pivotal mechanism to concentrate and compartmentalize rare molecules for forming primordial cells, ATP appears to control protein homeostasis and shape genome-proteome interfaces throughout the evolutionary trajectory, from prebiotic origins to modern cells.

Evolutionary analyses of intrinsically disordered regions reveal widespread signals of conservation.

Intrinsically disordered regions (IDRs) are segments of proteins without stable three-dimensional structures. As this flexibility allows them to interact with diverse binding partners, IDRs play key roles in cell signaling and gene expression. Despite the prevalence and importance of IDRs in eukaryotic proteomes and various biological processes, associating them with specific molecular functions remains a significant challenge due to their high rates of sequence evolution. However, by comparing the observed values of various IDR-associated properties against those generated under a simulated model of evolution, a recent study found most IDRs across the entire yeast proteome contain conserved features. Furthermore, it showed clusters of IDRs with common "evolutionary signatures," i.e. patterns of conserved features, were associated with specific biological functions. To determine if similar patterns of conservation are found in the IDRs of other systems, in this work we applied a series of phylogenetic models to over 7,500 orthologous IDRs identified in the Drosophila genome to dissect the forces driving their evolution. By comparing models of constrained and unconstrained continuous trait evolution using the Brownian motion and Ornstein-Uhlenbeck models, respectively, we identified signals of widespread constraint, indicating conservation of distributed features is mechanism of IDR evolution common to multiple biological systems. In contrast to the previous study in yeast, however, we observed limited evidence of IDR clusters with specific biological functions, which suggests a more complex relationship between evolutionary constraints and function in the IDRs of multicellular organisms.

Map conformational landscapes of intrinsically disordered proteins with polymer physics quantities.

Disordered proteins are conformationally flexible proteins that are biologically important and have been implicated in devastating diseases such as Alzheimer's disease and cancer. Unlike stably folded structured proteins, disordered proteins sample a range of different conformations that needs to be accounted for. Here, we treat disordered proteins as polymer chains, and compute a dimensionless quantity called instantaneous shape ratio (Rs), as Rs = Ree2/Rg2, where Ree is end-to-end distance and Rg is radius of gyration. Extended protein conformations tend to have high Ree compared with Rg, and thus have high Rs values, whereas compact conformations have smaller Rs values. We use a scatter plot of Rs (representing shape) against Rg (representing size) as a simple map of conformational landscapes. We first examine the conformational landscape of simple polymer models such as Random Walk, Self-Avoiding Walk, and Gaussian Walk (GW), and we notice that all protein/polymer maps lie within the boundaries of the GW map. We thus use the GW map as a reference and, to assess conformational diversity, we compute the fraction of the GW conformations (fC) covered by each protein/polymer. Disordered proteins all have high fC scores, consistent with their disordered nature. Each disordered protein accesses a different region of the reference map, revealing differences in their conformational ensembles. We additionally examine the conformational maps of the nonviral gene delivery vector polyethyleneimine at various protonation states, and find that they resemble disordered proteins, with coverage of the reference map decreasing with increasing protonation state, indicating decreasing conformational diversity. We propose that our method of combining Rs and Rg in a scatter plot generates a simple, meaningful map of the conformational landscape of a disordered protein, which in turn can be used to assess conformational diversity of disordered proteins.

Protein misfolding and amyloid nucleation through liquid-liquid phase separation.

Liquid-liquid phase separation (LLPS) is an emerging phenomenon in cell physiology and diseases. The weak multivalent interaction prerequisite for LLPS is believed to be facilitated through intrinsically disordered regions, which are prevalent in neurodegenerative disease-associated proteins. These aggregation-prone proteins also exhibit an inherent property for phase separation, resulting in protein-rich liquid-like droplets. The very high local protein concentration in the water-deficient confined microenvironment not only drives the viscoelastic transition from the liquid to solid-like state but also most often nucleate amyloid fibril formation. Indeed, protein misfolding, oligomerization, and amyloid aggregation are observed to be initiated from the LLPS of various neurodegeneration-related proteins. Moreover, in these cases, neurodegeneration-promoting genetic and environmental factors play a direct role in amyloid aggregation preceded by the phase separation. These cumulative recent observations ignite the possibility of LLPS being a prominent nucleation mechanism associated with aberrant protein aggregation. The present review elaborates on the nucleation mechanism of the amyloid aggregation pathway and the possible early molecular events associated with amyloid-related protein phase separation. It also summarizes the recent advancement in understanding the aberrant phase transition of major proteins contributing to neurodegeneration focusing on the common disease-associated factors. Overall, this review proposes a generic LLPS-mediated multistep nucleation mechanism for amyloid aggregation and its implication in neurodegeneration.

How an intrinsically disordered regulatory subunit assembles a PP1:eIF2 complex.

Fatalska et al.1 use an interdisciplinary strategy to elucidate how an intrinsically disordered regulatory subunit of protein phosphatase 1 binds trimeric eIF2 and positions the phosphatase-substrate complex for dephosphorylation. As validation, they show that a disease mutation abolishes the interaction.

Fuzzy recognition by the prokaryotic transcription factor HigA2 from Vibrio cholerae.

Disordered protein sequences can exhibit different binding modes, ranging from well-ordered folding-upon-binding to highly dynamic fuzzy binding. The primary function of the intrinsically disordered region of the antitoxin HigA2 from Vibrio cholerae is to neutralize HigB2 toxin through ultra-high-affinity folding-upon-binding interaction. Here, we show that the same intrinsically disordered region can also mediate fuzzy interactions with its operator DNA and, through interplay with the folded helix-turn-helix domain, regulates transcription from the higBA2 operon. NMR, SAXS, ITC and in vivo experiments converge towards a consistent picture where a specific set of residues in the intrinsically disordered region mediate electrostatic and hydrophobic interactions while "hovering" over the DNA operator. Sensitivity of the intrinsically disordered region to scrambling the sequence, position-specific contacts and absence of redundant, multivalent interactions, point towards a more specific type of fuzzy binding. Our work demonstrates how a bacterial regulator achieves dual functionality by utilizing two distinct interaction modes within the same disordered sequence.

Disordered clock protein interactions and charge blocks turn an hourglass into a persistent circadian oscillator.

Organismal physiology is widely regulated by the molecular circadian clock, a feedback loop composed of protein complexes whose members are enriched in intrinsically disordered regions. These regions can mediate protein-protein interactions via SLiMs, but the contribution of these disordered regions to clock protein interactions had not been elucidated. To determine the functionality of these disordered regions, we applied a synthetic peptide microarray approach to the disordered clock protein FRQ in Neurospora crassa. We identified residues required for FRQ's interaction with its partner protein FRH, the mutation of which demonstrated FRH is necessary for persistent clock oscillations but not repression of transcriptional activity. Additionally, the microarray demonstrated an enrichment of FRH binding to FRQ peptides with a net positive charge. We found that positively charged residues occurred in significant "blocks" within the amino acid sequence of FRQ and that ablation of one of these blocks affected both core clock timing and physiological clock output. Finally, we found positive charge clusters were a commonly shared molecular feature in repressive circadian clock proteins. Overall, our study suggests a mechanistic purpose for positive charge blocks and yielded insights into repressive arm protein roles in clock function.

Intrinsically disordered regions (IDRs): A vague and confusing concept for protein function.

The term "intrinsically disordered region" (IDR) in proteins has been used in numerous publications. However, most proteins contain IDRs, the term refers to very different types of structures and functions, and many IDRs become structured upon interaction with other biomolecules. Thus, IDR is an unnecessary, vague, and ultimately confusing concept.

Quantum Defect Sensitization via Phase-Changing Supercharged Antibody Fragments.

Quantum defects in single-walled carbon nanotubes promote exciton localization, which enables potential applications in biodevices and quantum light sources. However, the effects of local electric fields on the emissive energy states of quantum defects and how they can be controlled are unexplored. Here, we investigate quantum defect sensitization by engineering an intrinsically disordered protein to undergo a phase change at a quantum defect site. We designed a supercharged single-chain antibody fragment (scFv) to enable a full ligand-induced folding transition from an intrinsically disordered state to a compact folded state in the presence of a cytokine. The supercharged scFv was conjugated to a quantum defect to induce a substantial local electric change upon ligand binding. Employing the detection of a proinflammatory biomarker, interleukin-6, as a representative model system, supercharged scFv-coupled quantum defects exhibited robust fluorescence wavelength shifts concomitant with the protein folding transition. Quantum chemical simulations suggest that the quantum defects amplify the optical response to the localization of charges produced upon the antigen-induced folding of the proteins, which is difficult to achieve in unmodified nanotubes. These findings portend new approaches to modulate quantum defect emission for biomarker sensing and protein biophysics and to engineer proteins to modulate binding signal transduction.