Unstructured Region Research Articles

Snapshots of actin and tubulin folding inside the TRiC chaperonin

The integrity of a cell’s proteome depends on correct folding of polypeptides by chaperonins. The chaperonin TCP-1 ring complex (TRiC) acts as obligate folder for >10% of cytosolic proteins, including he cytoskeletal proteins actin and tubulin. Although its architecture and how it recognizes folding substrates are emerging from structural studies, the subsequent fate of substrates inside the TRiC chamber is not defined. We trapped endogenous human TRiC with substrates (actin, tubulin) and cochaperone (PhLP2A) at different folding stages, for structure determination by cryo-EM. The already-folded regions of client proteins are anchored at the chamber wall, positioning unstructured regions toward the central space to achieve their native fold. Substrates engage with different sections of the chamber during the folding cycle, coupled to TRiC open-and-close transitions. Further, the cochaperone PhLP2A modulates folding, acting as a molecular strut between substrate and TRiC chamber. Our structural snapshots piece together an emerging model of client protein folding within TRiC.

Control of Nucleophile Chemoselectivity in Cyanobactin YcaO Heterocyclases PatD and TruD.

Members of the YcaO superfamily are among the most common post-translational modification enzymes in natural product biosynthesis, with wide usage in biotechnology and synthetic biology applications. Here, we use domain-swapped chimeras and discovered unstructured regions in cyanobactin YcaOs that guide interactions with the substrates, governing access to interior amino acids in the substrates and explaining the chemoselectivity between PatD and TruD. These results define how the cyanobactin heterocyclases modify exceptionally sequence diverse substrates, yet with a high degree of positional and nucleophile selectivity.

The peroxisomal exportomer directly inhibits phosphoactivation of the pexophagy receptor Atg36 to suppress pexophagy in yeast.

Autophagy receptor (or adaptor) proteins facilitate lysosomal destruction of various organelles in response to cellular stress, including nutrient deprivation. To what extent membrane-resident autophagy receptors also respond to organelle-restricted cues to induce selective autophagy remains poorly understood. We find that latent activation of the yeast pexophagy receptor Atg36 by the casein kinase Hrr25 in rich media is repressed by the ATPase activity of Pex1/6, the catalytic subunits of the exportomer AAA+ transmembrane complex enabling protein import into peroxisomes. Quantitative proteomics of purified Pex3, an obligate Atg36 coreceptor, support a model in which the exportomer tail anchored to the peroxisome membrane represses Atg36 phosphorylation on Pex3 without assistance from additional membrane factors. Indeed, we reconstitute inhibition of Atg36 phosphorylation in vitro using soluble Pex1/6 and define an N-terminal unstructured region of Atg36 that enables regulation by binding to Pex1. Our findings uncover a mechanism by which a compartment-specific AAA+ complex mediating organelle biogenesis and protein quality control staves off induction of selective autophagy.

Investigations of pH-dependent dynamic properties of OmpG-16SL, an outer membrane protein G mutant by ATR-FTIR spectroscopy

In this paper, the dynamic properties of outer membrane protein G mutant (OmpG-16SL) are investigated with ATR-FTIR spectroscopy. While OmpG-WT has 14 β-strands in its structure, the mutant is designed to have 16 β-strands with the intention of creating an enlarged pore. Loop L6 is elongated by introducing six residues, two of which are negatively charged. The solvent accessibility of the OmpG-16SL mutant is compared with WT and a previously reported mutant OmpG-16S by tracking the 1H/2H exchange kinetics in acidic and neutral buffer conditions. The exchange kinetics and dynamics in the fast and slow exchange phases are separately investigated using the 2DCOS technique, which enables the tracking of the structural changes at each phase of the exchange process. The results suggest that the mutant OmpG-16SL is equally exposed to buffer in both acidic and neutral pH conditions. Additionally, the time range in the fast phase is very short - one-tenth of that for WT - and most of the exchange is completed in this phase. This fast exchange within minutes is also indicative of the presence of highly flexible and/or unstructured regions. In all, the fast exchange rates independent of the buffer pH justify the assumption that there is an altered interaction among the charged residues, which leads to a steadily-open pore. The role of the side-chain interactions within the pore and between the loops involving the loop L6 is also discussed.

Activation mechanism of the class D fungal GPCR dimer Ste2

The fungal class D1 G-protein-coupled receptor (GPCR) Ste2 has a different arrangement of transmembrane helices compared with mammalian GPCRs and a distinct mode of coupling to the heterotrimeric G protein Gpa1–Ste2–Ste181. In addition, Ste2 lacks conserved sequence motifs such as DRY, PIF and NPXXY, which are associated with the activation of class A GPCRs2. This suggested that the activation mechanism of Ste2 may also differ. Here we determined structures of Saccharomyces cerevisiae Ste2 in the absence of G protein in two different conformations bound to the native agonist α-factor, bound to an antagonist and without ligand. These structures revealed that Ste2 is indeed activated differently from other GPCRs. In the inactive state, the cytoplasmic end of transmembrane helix H7 is unstructured and packs between helices H1–H6, blocking the G protein coupling site. Agonist binding results in the outward movement of the extracellular ends of H6 and H7 by 6 Å. On the intracellular surface, the G protein coupling site is formed by a 20 Å outward movement of the unstructured region in H7 that unblocks the site, and a 12 Å inward movement of H6. This is a distinct mechanism in GPCRs, in which the movement of H6 and H7 upon agonist binding facilitates G protein coupling.

EP183: Functional assessment of a novel POU4F3 missense variant

The POU-domain family of transcription factors have a wide variety of functions and are critical regulators of many developmental processes. They are characterized by a common bipartite DNA-binding structure with POU-specific and POU-homeodomain subdomains, joined by an unstructured linkage region. Both subdomains are required for high affinity DNA binding of POU transcription factors. The POU4F3 gene has been found to be essential for inner-ear hair cell maintenance and is associated with autosomal-dominant nonsyndromic hearing loss with variable age of onset. Here we describe an individual with a novel heterozygous missense variant in POU4F3 exhibiting bilateral childhood onset sensorineural hearing loss. The proband’s mother also exhibited hearing loss. Functional assessment of this variant was performed to determine the impact on protein localization, and familial segregation studies were performed to better understand the pathogenicity of this variant. The proband underwent exome sequencing (ES) which revealed a maternally inherited missense variant in POU4F3 (NM_002700.2, c.856A>G, p.(Lys286Glu)). Family members were subsequently given audiograms and tested for this variant. To functionally assess the variant HA-tagged wild-type and mutant POU4F3 were expressed by using the mammalian expression vector pcDNA3.1 and transfected in Hek293 cells. To determine the subcellular localization of the HA-tagged wild-type and mutant POU4F3, transiently transfected cells were fixed with 4% paraformaldehyde, incubated with primary anti-HA and secondary goat-anti-mouse Alexa488-labelled antibodies. Images were taken with a fluorescence microscope and processed using ImageJ. To determine the number of cells expressing POU4F3 outside the nucleus, slides were blinded and 100 cells of each transfected mutant were scored; each construct was transfected in duplicate. Audiograms revealed that one of the proband’s brothers had mild hearing loss, while the other was unaffected. Familial variant testing then showed that the affected brother is heterozygous for the POU4F3 variant while the unaffected brother does not harbor the variant. The mother was previously shown to have hearing loss, and the variant was maternally inherited. Functional assessment of the variant demonstrated that it results in significant mis-localization from the nucleus to the cytoplasm compared to wild type controls (p value: 0.009). This affect was more significant than positive controls included in the analysis. The proband’s variant is not reported in population databases and is predicted to be deleterious by REVEL (0.89) and CADD (27.7). This variant was clinically classified as a VUS, with the following criteria applied: PM2+PP3. This variant falls within the homeodomain of the protein, which is required for DNA binding of POU transcription factors. Pathogenic missense variants in POU4F3 have previously been shown to disrupt the nuclear localization of the protein, so this was assessed in cells expressing the p.(Lys286Glu) variant. The proband’s variant was found to result in significant mis-localization of POU4F3 to the cytoplasm, suggesting an impact on the protein function. Finally, the variant was found to segregate with hearing loss in the proband’s mother and affected brother, allowing us to apply PP1 criteria based on the ClinGen hearing loss evaluation guidelines. Taken together these data are suggestive that this variant is causative for the proband, however this remains a VUS.

Influenza A Virus NS1 Protein Structural Flexibility Analysis According to Its Structural Polymorphism Using Computational Approaches

Influenza A viruses are highly contagious RNA viruses that cause respiratory tract infections in humans and animals. Their non-structural protein NS1, a homodimer of two 230-residue chains, is the main viral factor in counteracting the antiviral defenses of the host cell. Its RNA-binding domain is an obligate dimer that is connected to each of the two effector domains by a highly flexible unstructured linker region of ten amino acids. The flexibility of NS1 is a key property that allows its effector domains and its RNA binding domain to interact with several protein partners or RNAs. The three-dimensional structures of full-length NS1 dimers revealed that the effector domains could adopt three distinct conformations as regards their mutual interactions and their orientation relative to the RNA binding domain (closed, semi-open and open). The origin of this structural polymorphism is currently being investigated and several hypotheses are proposed, among which one posits that it is a strain-specific property. In the present study, we explored through computational molecular modeling the dynamic and flexibility properties of NS1 from three important influenza virus A strains belonging to three distinct subtypes (H1N1, H6N6, H5N1), for which at least one conformation is available in the Protein Data Bank. In order to verify whether NS1 is stable in three forms for the three strains, we constructed homology models if the corresponding forms were not available in the Protein Data Bank. Molecular dynamics simulations were performed in order to predict the stability over time of the three distinct sequence variants of NS1, in each of their three distinct conformations. Our results favor the co-existence of three stable structural forms, regardless of the strain, but also suggest that the length of the linker, along with the presence of specific amino acids, modulate the dynamic properties and the flexibility of NS1.

DNA-induced structural changes in DNA-damage suppressor protein Dsup by NMR and circular dichroism

DNA damage suppressor protein (Dsup) from water bear Ramazzottius varieormatus can suppress X-ray-induced DNA damage and improve radio-tolerance in human cells [Hashimoto, et al. Nat Commun 7, 12808 (2016)]. The functional information about this unique protein is very limited and its structure is unknown. Sequence analysis suggests that Dsup is an intrinsically disordered protein with extended unstructured regions and a potential single-helix domain. We are studying Dsup using a variety of biophysical methods to understand the basis of its interaction with DNA and the mechanism of DNA protection.

SIRT2 regulates proliferation and chemotherapy response of MLL-ENL-driven acute myeloid leukemia

Both MLL-AF9 and MLL-ENL leukemia fusion proteins drive oncogenic transformation of hematopoietic cells through their N-terminal DNA/histone binding mixed-lineage leukemia 1 domain and C-terminal fragment of AF9 or ENL containing an unstructured linker region and the ANC1 homology domain, which recruits transcription factors. Despite of their structural similarity, acute myeloid leukemia (AML) patients bearing MLL-ENL show more adverse outcomes compared to those with MLL-AF9. We recapitulated the clinical patterns of these two MLL-fusions driven AMLs using murine models and found that MLL-ENL AML cells showed slower cell cycle progression and more resistance to standard chemotherapy than MLL-AF9 cells. These phenotypes were primarily controlled by the linker regions of ENL and a highly conserved lysine residue K469 within. Substitution of K469 with an acetylated mimic glutamine abolished the ability of MLL-ENL to suppress proliferation and promote chemo-resistance. We showed that deacetylase Sirt2 might act as an upstream regulator of MLL-ENL. Deletion of Sirt2 promoted proliferation of AML cells with either MLL fusions. Importantly, loss of Sirt2 greatly enhanced the sensitivity of the MLL-ENL AML cells to chemo-treatment. Taken together, our study uncovered a unique regulatory role of Sirt2 in leukemogenesis and suggested targeting SIRT2 as a new way to sensitize MLL-ENL AML patience for chemotherapy.

Proteome‐scale mapping of binding sites in the unstructured regions of the human proteome

Specific protein–protein interactions are central to all processes that underlie cell physiology. Numerous studies have together identified hundreds of thousands of human protein–protein interactions. However, many interactions remain to be discovered, and low affinity, conditional, and cell type‐specific interactions are likely to be disproportionately underrepresented. Here, we describe an optimized proteomic peptide‐phage display library that tiles all disordered regions of the human proteome and allows the screening of ~ 1,000,000 overlapping peptides in a single binding assay. We define guidelines for processing, filtering, and ranking the results and provide PepTools, a toolkit to annotate the identified hits. We uncovered >2,000 interaction pairs for 35 known short linear motif (SLiM)‐binding domains and confirmed the quality of the produced data by complementary biophysical or cell‐based assays. Finally, we show how the amino acid resolution‐binding site information can be used to pinpoint functionally important disease mutations and phosphorylation events in intrinsically disordered regions of the proteome. The optimized human disorderome library paired with PepTools represents a powerful pipeline for unbiased proteome‐wide discovery of SLiM‐based interactions.

A quinoline alkaloid potentially modulates the amyloidogenic structural transitions of the biofilm scaffolding small basic protein

Bacterial biofilm formation by communities of opportunistic bacterial pathogens like Staphylococcus epidermidis is regarded as the primary virulence mechanism facilitating the spread of detrimental nosocomial and implant-associated infections. An 18-kDa small basic protein (Sbp) and its amyloid fibrils account for strengthening the biofilm architecture and scaffolding the S. epidermidis biofilm matrix. Our study reports systematic analysis of the amyloidogenic structural transitions of Sbp and predicts the amyloid core of the protein which may trigger misfolding and aggregation. Herein, we report the novel amyloid inhibitory potential of Camptothecin, a quinoline alkaloid which binds stably to Sbp monomers and redirects the formation of unstructured regions further destabilizing the protein. Molecular dynamics simulations reveal that Camptothecin averts β-sheet transitions, interrupts with electrostatic interactions and disrupts the intermolecular hydrophobic associations between the exposed hydrophobic amyloidogenic regions of Sbp. Collectively, our study puts forward the first report detailing the heteromolecular associations and amyloid modulatory effects of Camptothecin which may serve as a structural scaffold for the tailored designing of novel drugs targeting the S. epidermidis biofilm matrix. Communicated by Ramaswamy H. Sarma

Fission yeast Ase1PRC1 is required for the G2-microtubule damage response.

Schizosaccharomyces pombe delays entry into mitosis following G2 microtubule damage. This pathway is dependent on Rad26ATRIP, the regulatory subunit of the Rad26ATRIP/Rad3ATR DNA damage response (DDR) complex. However, this G2 microtubule damage response pathway acts independently of the G2 DNA damage checkpoint pathway. To identify other proteins in this G2 microtubule damage pathway, we previously screened a cDNA overexpression library for genes that rescued the sensitivity of rad26Δ cells to the microtubule poison thiabendazole. A partial cDNA fragment encoding only the C-terminal regulatory region of the microtubule bundling protein Ase1PRC1 was isolated. This fragment lacks the Ase1PRC1 dimerization and microtubule binding domains and retains the conserved C-terminal unstructured regulatory region. Here, we report that ase1Δ cells fail to delay entry into mitosis following G2 microtubule damage. Microscopy revealed that Rad26ATRIP foci localized alongside Ase1PRC1 filaments, although we suggest that this is related to microtubule-dependent double strand break mobility that facilitates homologous recombination events. Indeed, we report that the DNA repair protein Rad52 co-localizes with Rad26ATRIP at these foci, and that localization of Rad26ATRIP to these foci depends on a Rad26ATRIP N-terminal region containing a checkpoint recruitment domain. To our knowledge, this is the first report implicating Ase1PRC1 in regulation of the G2/M transition.

A ubiquitous disordered protein interaction module orchestrates transcription elongation.

During eukaryotic transcription elongation, RNA polymerase II (RNAP2) is regulated by a chorus of factors. Here, we identified a common binary interaction module consisting of TFIIS N-terminal domains (TNDs) and natively unstructured TND-interacting motifs (TIMs). This module was conserved among the elongation machinery and linked complexes including transcription factor TFIIS, Mediator, super elongation complex, elongin, IWS1, SPT6, PP1-PNUTS phosphatase, H3K36me3 readers, and other factors. Using nuclear magnetic resonance, live-cell microscopy, and mass spectrometry, we revealed the structural basis for these interactions and found that TND-TIM sequences were necessary and sufficient to induce strong and specific colocalization in the crowded nuclear environment. Disruption of a single TIM in IWS1 induced robust changes in gene expression and RNAP2 elongation dynamics, which underscores the functional importance of TND-TIM surfaces for transcription elongation.

Mechanisms of CP190 Interaction with Architectural Proteins in Drosophila Melanogaster.

Most of the known Drosophila architectural proteins interact with an important cofactor, CP190, that contains three domains (BTB, M, and D) that are involved in protein–protein interactions. The highly conserved N-terminal CP190 BTB domain forms a stable homodimer that interacts with unstructured regions in the three best-characterized architectural proteins: dCTCF, Su(Hw), and Pita. Here, we identified two new CP190 partners, CG4730 and CG31365, that interact with the BTB domain. The CP190 BTB resembles the previously characterized human BCL6 BTB domain, which uses its hydrophobic groove to specifically associate with unstructured regions of several transcriptional repressors. Using GST pull-down and yeast two-hybrid assays, we demonstrated that mutations in the hydrophobic groove strongly affect the affinity of CP190 BTB for the architectural proteins. In the yeast two-hybrid assay, we found that architectural proteins use various mechanisms to improve the efficiency of interaction with CP190. Pita and Su(Hw) have two unstructured regions that appear to simultaneously interact with hydrophobic grooves in the BTB dimer. In dCTCF and CG31365, two adjacent regions interact simultaneously with the hydrophobic groove of the BTB and the M domain of CP190. Finally, CG4730 interacts with the BTB, M, and D domains of CP190 simultaneously. These results suggest that architectural proteins use different mechanisms to increase the efficiency of interaction with CP190.

Folding Steps in the Fibrillation of Functional Amyloid: Denaturant Sensitivity Reveals Common Features in Nucleation and Elongation

Functional bacterial amyloids (FuBA) are intrinsically disordered proteins (IDPs) which rapidly and efficiently aggregate, forming extremely stable fibrils. The conversion from IDP to amyloid is evolutionarily optimized and likely couples folding to association. Many FuBA contain several imperfect repeat sequences which contribute to the stability of mature FuBA fibrils. Aggregation can be considered an intermolecular extension of the process of intramolecular protein folding which has traditionally been studied using chemical denaturants. Here we employ denaturants to investigate folding steps during fibrillation of CsgA and FapC. We quantify protein compactification (i.e. the extent of burial of otherwise exposed surface area upon association of proteins) during different stages of fibrillation based on the dependence of fibrillation rate constants on the denaturant concentration (m-values) determined from fibrillation curves. For both proteins, urea mainly affects nucleation and elongation (not fragmentation), consistent with the fact that these steps involve both intra- and intermolecular association. The two steps have similar m-values, indicating that activation steps in nucleation and elongation involve the same level of folding. Surprisingly, deletion of two or three repeats from FapC leads to larger m-values (i.e. higher compactification) during the activation step of fibril growth. This observation is extended by SAXS analysis of the fibrils which indicates that weakening of the amyloidogenic core caused by repeat deletions causes a larger portion of normally unstructured regions of the protein to be included into the amyloid backbone. We conclude that the sensitivity of fibrillation to denaturants can provide useful insight into molecular mechanisms of aggregation.

Man does not live by intrinsically unstructured proteins alone: The role of structured regions in aggregation.

Protein misfolding is a topic that is of primary interest both in biology and medicine because of its impact on fundamental processes and disease. In this review, we revisit the concept of protein misfolding and discuss how the field has evolved from the study of globular folded proteins to focusing mainly on intrinsically unstructured and often disordered regions. We argue that this shift of paradigm reflects the more recent realisation that misfolding may not only be an adverse event, as originally considered, but also may fulfil a basic biological need to compartmentalise the cell with transient reversible granules. We nevertheless provide examples in which structure is an important component of a much more complex aggregation behaviour that involves both structured and unstructured regions of a protein. We thus suggest that a more comprehensive evaluation of the mechanisms that lead to aggregation might be necessary.

Quadratic splines on quad-tri meshes: Construction and an application to simulations on watertight reconstructions of trimmed surfaces

Given an unstructured mesh consisting of quadrilaterals and triangles (we allow both planar and non-planar meshes of arbitrary topology), we present the construction of quadratic splines of mixed smoothness — C1 smooth away from the unstructured regions of T and C0 smooth otherwise. The splines have several useful B-spline-like properties – partition of unity, non-negativity, local support and linear independence – and allow for straightforward imposition of boundary conditions. We propose a non-nested refinement process for the splines with multiple advantages — a simple computer implementation, reduction in the footprint of C0 smoothness, boundary preservation, and excellent approximation behaviour in simulations. Furthermore, the refinement process leaves the splines invariant on the mesh boundary. Numerical tests indicate that the spline spaces demonstrate optimal approximation behaviour in the L2 and H1 norms under mesh refinement, and provide a viable approach to simulations on watertight reconstructions of trimmed surfaces.

Role of “dual-personality” fragments in HEV adaptation—analysis of Y-domain region

BackgroundHepatitis E is a liver disease caused by the pathogen hepatitis E virus (HEV). The largest polyprotein open reading frame 1 (ORF1) contains a nonstructural Y-domain region (YDR) whose activity in HEV adaptation remains uncharted. The specific role of disordered regions in several nonstructural proteins has been demonstrated to participate in the multiplication and multiple regulatory functions of the viruses. Thus, intrinsic disorder of YDR including its structural and functional annotation was comprehensively studied by exploiting computational methodologies to delineate its role in viral adaptation. ResultsBased on our findings, it was evident that YDR contains significantly higher levels of ordered regions with less prevalence of disordered residues. Sequence-based analysis of YDR revealed it as a “dual personality” (DP) protein due to the presence of both structured and unstructured (intrinsically disordered) regions. The evolution of YDR was shaped by pressures that lead towards predominance of both disordered and regularly folded amino acids (Ala, Arg, Gly, Ile, Leu, Phe, Pro, Ser, Tyr, Val). Additionally, the predominance of characteristic DP residues (Thr, Arg, Gly, and Pro) further showed the order as well as disorder characteristic possessed by YDR. The intrinsic disorder propensity analysis of YDR revealed it as a moderately disordered protein. All the YDR sequences consisted of molecular recognition features (MoRFs), i.e., intrinsic disorder-based protein–protein interaction (PPI) sites, in addition to several nucleotide-binding sites. Thus, the presence of molecular recognition (PPI, RNA binding, and DNA binding) signifies the YDR’s interaction with specific partners, host membranes leading to further viral infection. The presence of various disordered-based phosphorylation sites further signifies the role of YDR in various biological processes. Furthermore, functional annotation of YDR revealed it as a multifunctional-associated protein, due to its susceptibility in binding to a wide range of ligands and involvement in various catalytic activities. ConclusionsAs DP are targets for regulation, thus, YDR contributes to cellular signaling processes through PPIs. As YDR is incompletely understood, therefore, our data on disorder-based function could help in better understanding its associated functions. Collectively, our novel data from this comprehensive investigation is the first attempt to delineate YDR role in the regulation and pathogenesis of HEV.

Informed Autonomous Exploration of Subterranean Environments

Autonomous exploration is highly challenging in subterranean applications due to the constraints imposed by the nature of the environments (e.g., dead-end branches, unstructured regions, narrow passages and bifurcations). Robots need to constantly balance their exploration objectives with measures to ensure safety. We present an informed exploration approach to address these challenges, which exploits a reachability graph to represent the environment's structure and adaptive navigation to find collision-free motions. Our system makes the inspection task tractable and maximizes the information acquired about the environment while preserving safety. We evaluate our navigation and exploration techniques against several challenging cave scenarios reconstructed using real data. Our experimental results demonstrate that our method enables the robot to make informed decisions and perform exploration more efficiently than existing techniques in confined spaces.

Evidence for an Ordering Transition near 120 K in an Intrinsically Disordered Protein, Casein.

Intrinsically disordered proteins (IDPs) are proteins that possess large unstructured regions. Their importance is increasingly recognized in biology but their characterization remains a challenging task. We employed field swept Electron Spin Echoes in pulsed EPR to investigate low-temperature stochastic molecular librations in a spin-labeled IDP, casein (the main protein of milk). For comparison, a spin-labeled globular protein, hen egg white lysozyme, is also investigated. For casein these motions were found to start at 100 K while for lysozyme only above 130 K, which was ascribed to a denser and more ordered molecular packing in lysozyme. However, above 120 K, the motions in casein were found to depend on temperature much slower than those in lysozyme. This abrupt change in casein was assigned to an ordering transition in which peptide residues rearrange making the molecular packing more rigid and/or more cohesive. The found features of molecular motions in these two proteins turned out to be very similar to those known for gel-phase lipid bilayers composed of conformationally ordered and conformationally disordered lipids. This analogy with a simpler molecular system may appear helpful for elucidation properties of molecular packing in IDPs.