Intrinsically Disordered Proteins Research Articles

Mapping Full Conformational Transition Dynamics of Intrinsically Disordered Proteins Using a Single-Molecule Nanocircuit.

Intrinsically disordered proteins (IDPs) are emerging therapeutic targets for human diseases. However, probing their transient conformations remains challenging because of conformational heterogeneity. To address this problem, we developed a biosensor using a point-functionalized silicon nanowire (SiNW) that allows for real-time sampling of single-molecule dynamics. A single IDP, N-terminal transactivation domain of tumor suppressor protein p53 (p53TAD1), was covalently conjugated to the SiNW through chemical engineering, and its conformational transition dynamics was characterized as current fluctuations. Furthermore, when a globular protein ligand in solution bound to the targeted p53TAD1, protein-protein interactions could be unambiguously distinguished from large-amplitude current signals. These proof-of-concept experiments enable semiquantitative, realistic characterization of the structural properties of IDPs and constitute the basis for developing a valuable tool for protein profiling and drug discovery in the future.

Multivalency drives interactions of alpha-synuclein fibrils with tau.

Age-related neurodegenerative disorders like Alzheimer's disease (AD) and Parkinson's disease (PD) are characterized by deposits of protein aggregates, or amyloid, in various regions of the brain. Historically, aggregation of a single protein was observed to be correlated with these different pathologies: tau in AD and α-synuclein (αS) in PD. However, there is increasing evidence that the pathologies of these two diseases overlap, and the individual proteins may even promote each other's aggregation. Both tau and αS are intrinsically disordered proteins (IDPs), lacking stable secondary and tertiary structure under physiological conditions. In this study we used a combination of biochemical and biophysical techniques to interrogate the interaction of tau with both soluble and fibrillar αS. Fluorescence correlation spectroscopy (FCS) was used to assess the interactions of specific domains of fluorescently labeled tau with full length and C-terminally truncated αS in both monomer and fibrillar forms. We found that full-length tau as well as individual tau domains interact with monomer αS weakly, but this interaction is much more pronounced with αS aggregates. αS aggregates also mildly slow the rate of tau aggregation, although not the final degree of aggregation. Our findings suggest that co-occurrence of tau and αS in disease are more likely to occur through monomer-fiber binding interactions, rather than monomer-monomer or co-aggregation.

A Few Charged Residues in Galectin-3's Folded and Disordered Regions Regulate Phase Separation.

Proteins with intrinsically disordered regions (IDRs) often undergo phase separation to control their functions spatiotemporally. Changing the pH alters the protonation levels of charged sidechains, which in turn affects the attractive or repulsive force for phase separation. In a cell, the rupture of membrane-bound compartments, such as lysosomes, creates an abrupt change in pH. However, how proteins' phase separation reacts to different pH environments remains largely unexplored. Here, using extensive mutagenesis, NMR spectroscopy, and biophysical techniques, it is shown that the assembly of galectin-3, a widely studied lysosomal damage marker, is driven by cation-π interactions between positively charged residues in its folded domain with aromatic residues in the IDR in addition to π-π interaction between IDRs. It is also found that the sole two negatively charged residues in its IDR sense pH changes for tuning the condensation tendency. Also, these two residues may prevent this prion-like IDR domain from forming rapid and extensive aggregates. These results demonstrate how cation-π, π-π, and electrostatic interactions can regulate protein condensation between disordered and structured domains and highlight the importance of sparse negatively charged residues in prion-like IDRs.

A pH-Dependent Coarse-Grained Model for Disordered Proteins: Histidine Interactions Modulate Conformational Ensembles.

Histidine (His) presents a unique challenge for modeling disordered protein conformations, as it is versatile and occurs in both the neutral (His0) and positively charged (His+) states. These His charge states, which are enabled by its imidazole side chain, influence the electrostatic and short-range interactions of His residues, which potentially engage in cation-π, π-π, and charge-charge interactions. Existing coarse-grained (CG) models often simplify His representation by assigning it an average charge, thereby neglecting these potential short-range interactions. To address this gap, we developed a model for intrinsically disordered proteins (IDPs) that accounts for the properties of histidine (H). The resulting IDPH model is a 21-amino acid CG model incorporating both His charge states. We show that interactions involving previously neglected His0 are critical for accurate modeling at high pH, where they significantly influence the compaction of His-rich IDPs such as Histatin-5 and CPEB4. These interactions contribute to structural stabilizations primarily via His0-His0 and His0-Arg interactions, which are overlooked in models focusing solely on the charged His+ state.

Conformational dependence of chemical shifts in the proline rich region of TAU protein.

Nuclear magnetic resonance (NMR) is an important method for structure elucidation of proteins, as it is an easily accessible and well understood method. To characterize intrinsically disordered proteins (IDPs) using computational models it is often necessary to analyze and integrate calculated observables with measurements derived from solution NMR experiments. In this case study, we investigate whether and which chemical shifts of the proline-rich region of Tau protein (residues 210-240) offer information about the conformational state to distinguish two different microscopic conformers. Using multiple computational methods, the chemical shifts of these two conformationally distinct structures are calculated. The different methods are compared regarding their ability to compute chemical shifts that are sensitive to conformational change. The analysis of the data shows significant differences between the available methods and gives suggestions for an improved pathway for ensemble reweighting. Nevertheless, the variation in the chemical shifts which are predicted for configurations that are commonly considered to belong to the same conformation is such that this obscures a comparison between distinct conformations. Conformational sensitivity is found for up to ∼26% of calculated chemical shifts. It is found to be unrelated to the atom element and has a minor relationship with the change in the corresponding ϕ dihedral angle.

Balancing thermodynamic stability, dynamics, and kinetics in phase separation of intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) are prevalent participants in liquid-liquid phase separation due to their inherent potential for promoting multivalent binding. Understanding the underlying mechanisms of phase separation is challenging, as phase separation is a complex process, involving numerous molecules and various types of interactions. Here, we used a simplified coarse-grained model of IDPs to investigate the thermodynamic stability of the dense phase, conformational properties of IDPs, chain dynamics, and kinetic rates of forming condensates. We focused on the IDP system, in which the oppositely charged IDPs are maximally segregated, inherently possessing a high propensity for phase separation. By varying interaction strengths, salt concentrations, and temperatures, we observed that IDPs in the dense phase exhibited highly conserved conformational characteristics, which are more extended than those in the dilute phase. Although the chain motions and global conformational dynamics of IDPs in the condensates are slow due to the high viscosity, local chain flexibility at the short timescales is largely preserved with respect to that at the free state. Strikingly, we observed a non-monotonic relationship between interaction strengths and kinetic rates for forming condensates. As strong interactions of IDPs result in high stable condensates, our results suggest that the thermodynamics and kinetics of phase separation are decoupled and optimized by the speed-stability balance through underlying molecular interactions. Our findings contribute to the molecular-level understanding of phase separation and offer valuable insights into the developments of engineering strategies for precise regulation of biomolecular condensates.

Positive Selection Drives the Evolution of the Structural Maintenance of Chromosomes (SMC) Complexes

Structural Maintenance of Chromosomes (SMC) complexes are an evolutionary conserved protein family. In most eukaryotes, three SMC complexes have been characterized, as follows: cohesin, condensin, and SMC5/6 complexes. These complexes are involved in a plethora of functions, and defects in SMC genes can lead to an increased risk of chromosomal abnormalities, infertility, and cancer. To investigate the evolution of SMC complex genes in mammals, we analyzed their selective patterns in an extended phylogeny. Signals of positive selection were identified for condensin NCAPG, for two SMC5/6 complex genes (SMC5 and NSMCE4A), and for all cohesin genes with almost exclusive meiotic expression (RAD21L1, REC8, SMC1B, and STAG3). For the latter, evolutionary rates correlate with expression during female meiosis, and most positively selected sites fall in intrinsically disordered regions (IDRs). Our results support growing evidence that IDRs are fast evolving, and that they most likely contribute to adaptation through modulation of phase separation. We suggest that the natural selection signals identified in SMC complexes may be the result of different selective pressures: a host-pathogen arms race in the condensin and SMC5/6 complexes, and an intragenomic conflict for meiotic cohesin genes that is similar to that described for centromeres and telomeres.

A land plant phylogenetic framework for GLABROUS INFLORESCENCE STEMS (GIS), SUPERMAN, JAGGED and allies plus their TOPLESS co-repressor

Members of the plant specific family of C1-1i zincfinger transcriptionfactors (ZF-TFs), such as SUPERMAN, JAGGED, KNUCKLES or GIS,regulatediversedevelopmental processes including sexual reproduction. C1-1is consist of one zinc-finger and one to two EAR domains, connected by large intrinsically disordered regions (IDR). While the role of C1-i1 ZF-TFs in development processes is well known for some genes in Arabidopsis, rice or tomatoa comprehensive and broadphylogenetic background is lacking, yet knowledge of orthology is a requirement for a better understanding of C1-1i-Zf-TFs diverse roles in plants. Here, we provide a fine-grained and land plant wide classification of C1-1i sub-families and their known co-repressors TOPLESS and TOPLESS RELATED. Our work combines the identification of orthologous groups with Maximum-Likelihood phylogeny reconstructions and digital gene expression analyses mining high quality land plant genomes and transcriptomes to generate a comprehensive framework of C1-1i ZF-TF evolution. We show that C1-1i’s are low to moderate copy genesand that orthologous genesonly partiallyhaveconserved sub-family and life cycle stage dependent expression pattern across land plants while others are highly diverged. Our workprovides the phylogenetic framework for C1-1i ZF-TFs, s and strengthen C1-1 ZF-TFs as a potential model for IDR-research in plants.

Design of intrinsically disordered protein variants with diverse structural properties.

Intrinsically disordered proteins (IDPs) perform a broad range of functions in biology, suggesting that the ability to design IDPs could help expand the repertoire of proteins with novel functions. Computational design of IDPs with specific conformational properties has, however, been difficult because of their substantial dynamics and structural complexity. We describe a general algorithm for designing IDPs with specific structural properties. We demonstrate the power of the algorithm by generating variants of naturally occurring IDPs that differ in compaction, long-range contacts, and propensity to phase separate. We experimentally tested and validated our designs and analyzed the sequence features that determine conformations. We show how our results are captured by a machine learning model, enabling us to speed up the algorithm. Our work expands the toolbox for computational protein design and will facilitate the design of proteins whose functions exploit the many properties afforded by protein disorder.

Genetically-encoded phase separation sensors for intracellular probing of biomolecular condensates.

Biomolecular condensates are dynamic membraneless compartments with enigmatic roles across intracellular phenomena. Intrinsically-disordered proteins (IDPs) often function as condensate scaffolds, fueled by their liquid-liquid phase separation (LLPS) dynamics. Intracellular probing of these condensates relies on live-cell imaging of IDP-scaffolds tagged with fluorescent proteins. Conformational heterogeneity in IDPs, however, renders them uniquely sensitive to molecular-level fusions, risking distortion of the native biophysical properties of IDP-scaffolds and their assemblies. Probing epidermal condensates in mouse skin, we recently introduced genetically encoded LLPS-sensors that circumvent the need for molecular-level tagging of skin IDPs. The concept of LLPS-sensors involves a shift in focus from subcellular tracking of IDP-scaffolds to higher-level observations that report on the assembly and liquid-dynamics of their condensates. Towards advancing the repertoire of intracellular LLPS-sensors, here we demonstrate biomolecular approaches for the evolution and tunability of epidermal LLPS-sensors and assess their impact in early and late stages of intracellular LLPS dynamics. Benchmarking against scaffold-bound fluorescent reporters, we found that tunable ultraweak scaffold-sensor interactions are key to the sensitive and innocuous probing of nascent and established biomolecular condensates. Our LLPS-sensitive tools pave the way for the high-fidelity intracellular probing of IDP-governed biomolecular condensates across biological systems.

Intrinsic Disorder and Other Malleable Arsenals of Evolved Protein Multifunctionality.

Microscopic evolution at the functional biomolecular level is an ongoing process. Leveraging functional and high-throughput assays, along with computational data mining, has led to a remarkable expansion of our understanding of multifunctional protein (and gene) families over the past few decades. Various molecular and intermolecular mechanisms are now known that collectively meet the cumulative multifunctional demands in higher organisms along an evolutionary path. This multitasking ability is attributed to a certain degree of intrinsic or adapted flexibility at the structure-function level. Evolutionary diversification of structure-function relationships in proteins highlights the functional importance of intrinsically disordered proteins/regions (IDPs/IDRs) which are highly dynamic biological soft matter. Multifunctionality is favorably supported by the fluid-like shapes of IDPs/IDRs, enabling them to undergo disorder-to-order transitions upon binding to different molecular partners. Other new malleable members of the protein superfamily, such as those involved in fold-switching, also undergo structural transitions. This new insight diverges from all traditional notions of functional singularity in enzyme classes and emphasizes a far more complex, multi-layered diversification of protein functionality. However, a thorough review in this line, focusing on flexibility and function-driven structural transitions related to evolved multifunctionality in proteins, is currently missing. This review attempts to address this gap while broadening the scope of multifunctionality beyond single protein sequences. It argues that protein intrinsic disorder is likely the most striking mechanism for expressing multifunctionality in proteins. A phenomenological analogy has also been drawn to illustrate the increasingly complex nature of modern digital life, driven by the need for multitasking, particularly involving media.

Evolution of a Eukaryotic Transcription Factor's co-TF Dependence Involves Multiple Intrinsically Disordered Regions Affecting Activation and Autoinhibition.

Combinatorial control by multiple transcription factors (TFs) is a hallmark of eukaryotic gene regulation. Despite its prevalence and crucial roles in enhancing specificity and integrating information, the mechanisms behind why eukaryotic TFs depend on one another, and whether such interdependence evolves, are not well understood. We exploit natural variation in co-TF dependence in the yeast phosphate starvation (PHO) response to address this question. In the model yeast Saccharomyces cerevisiae , the main TF, Pho4, relies on the co-TF Pho2 to regulate ∼28 genes. In a related yeast pathogen, Candida glabrata , its Pho4 exhibits significantly reduced Pho2 dependence and has an expanded target set of ∼70 genes. Biochemical analyses showed C. glabrata Pho4 (CgPho4) binds to the same consensus motif with 3-4-fold higher affinity than ScPho4 does. A machine-learning-based prediction and yeast one-hybrid assay identified two Intrinsically Disordered Regions (IDRs) in CgPho4 that boost the activity of the main activation domain but showed little to no activity on their own. We also found evidence for autoinhibition behind the co-TF dependence in ScPho4. An IDR in ScPho4 next to its DNA binding domain was found to act as a double-edged sword: it both allows for enhanced activity with Pho2, and inhibits Pho4's activity without Pho2. This study provides a detailed molecular picture of how co-TF dependence is mediated and how its evolution, mainly driven by IDR divergence, can lead to significant rewiring of the regulatory network.

α-Crystalline Domains and Intrinsically Disordered Regions Can Work in Parallel to Induce Accumulation of MBD6 at Chromocenters in Arabidopsis thaliana

Proteins are localized and concentrated at cellular and genomic locations for specific and efficient functions. Efforts to understand protein accumulation in eukaryotic organisms have primarily focused on multivalent interactions between intrinsically disordered regions (IDRs) as mediators of protein condensation. We previously showed that α-crystalline domain (ACD) proteins 15 (ACD15) and 21 (ACD21) were required for multimerization and the accumulation of gene-silencing methyl-CpG-binding domain protein 6 (MBD6) at chromocenters in Arabidopsis thaliana. Here, we demonstrate that ACDs and IDRs can act as parallel mechanisms, facilitating higher-order MBD6 assemblies. Using human IDRs known to be important for protein accumulation, we replicated and enhanced the accumulation of MBD6 at chromocenters. In addition, IDRs fused to MBD6 could substitute for ACD function and partially reconstitute the MBD6 gene-silencing function. However, the accumulation of MBD6 by IDRs still required ACD15 and ACD21 for full effect. These results establish that ACD-mediated protein accumulation is a mechanism that can function similarly to and together with IDR-mediated mechanisms.

Beyond monopole electrostatics in regulating conformations of intrinsically disordered proteins.

Conformations and dynamics of an intrinsically disordered protein (IDP) depend on its composition of charged and uncharged amino acids, and their specific placement in the protein sequence. In general, the charge (positive or negative) on an amino acid residue in the protein is not a fixed quantity. Each of the ionizable groups can exist in an equilibrated distribution of fully ionized state (monopole) and an ion-pair (dipole) state formed between the ionizing group and its counterion from the background electrolyte solution. The dipole formation (counterion condensation) depends on the protein conformation, which in turn depends on the distribution of charges and dipoles on the molecule. Consequently, effective charges of ionizable groups in the IDP backbone may differ from their chemical charges in isolation-a phenomenon termed charge-regulation. Accounting for the inevitable dipolar interactions, that have so far been ignored, and using a self-consistent procedure, we present a theory of charge-regulation as a function of sequence, temperature, and ionic strength. The theory quantitatively agrees with both charge reduction and salt-dependent conformation data of Prothymosin-alpha and makes several testable predictions. We predict charged groups are less ionized in sequences where opposite charges are well mixed compared to sequences where they are strongly segregated. Emergence of dipolar interactions from charge-regulation allows spontaneous coexistence of two phases having different conformations and charge states, sensitively depending on the charge patterning. These findings highlight sequence dependent charge-regulation and its potential exploitation by biological regulators such as phosphorylation and mutations in controlling protein conformation and function.

StrIDR: a database of intrinsically disordered regions of proteins with experimentally resolved structures.

Intrinsically disordered regions (IDRs) of proteins exist as an ensemble of conformations, and not as a single structure. Existing databases contain extensive, experimentally derived annotations of intrinsic disorder for millions of proteins at the sequence level. However, only a tiny fraction of these IDRs are associated with an experimentally determined protein structure. Moreover, even if a structure exists, parts of the disordered regions may still be unresolved. Here we organize Structures of Intrinsically Disordered Regions (StrIDR), a database of IDRs confirmed via experimental or homology-based evidence, resolved in experimentally determined structures. The database can provide useful insights into the dynamics, folding, and interactions of IDRs. It can also facilitate computational studies on IDRs, such as those using molecular dynamics simulations and/or machine learning. StrIDR is available at https://isblab.ncbs.res.in/stridr. The web UI allows for downloading PDB structures and SIFTS mappings of individual entries. Additionally, the entire database can be downloaded in a JSON format. The source code for creating and updating the database is available at https://github.com/isblab/stridr.

Identification of an intrinsically disordered region (IDR) in arginyltransferase 1 (ATE1).

Arginyltransferase 1 (ATE1) catalyzes arginylation, an important post-translational modification (PTM) in eukaryotes that plays a critical role in cellular homeostasis. The disruption of ATE1 function is implicated in mammalian neurodegenerative disorders and cardiovascular maldevelopment, while post-translational arginylation has also been linked to the activities of several important human viruses such as SARS-CoV-2 and HIV. Despite the known significance of ATE1 in mammalian cellular function, past biophysical studies of this enzyme have mainly focused on yeast ATE1, leaving the mechanism of arginylation in mammalian cells unclear. In this study, we sought to structurally and biophysically characterize mouse (Mus musculus) ATE1. Using size-exclusion chromatography (SEC), small angle X-ray scattering (SAXS), and hydrogen deuterium exchange mass spectrometry (HDX-MS), assisted by AlphaFold modeling, we found that mouse ATE1 is structurally more complex than yeast ATE1. Importantly, our data indicate the existence of an intrinsically disordered region (IDR) in all mouse ATE1 splice variants. However, comparative HDX-MS analyses show that yeast ATE1 does not have such an IDR, consistent with prior X-ray, cryo-EM, and SAXS analyses. Furthermore, bioinformatics approaches reveal that mammalian ATE1 sequences, as well as in a large majority of other eukaryotes, contain an IDR-like sequence positioned in proximity to the ATE1 GNAT active-site fold. Computational analysis suggests that the IDR likely facilitates the formation of the complex between ATE1 and tRNAArg, adding a new complexity to ATE1 structure and providing new insights for future studies of ATE1 functions.

Specific multivalent molecules boost CRISPR-mediated transcriptional activation

CRISPR/Cas-based transcriptional activators can be enhanced by intrinsically disordered regions (IDRs). However, the underlying mechanisms are still debatable. Here, we examine 12 well-known IDRs by fusing them to the dCas9-VP64 activator, of which only seven can augment activation, albeit independently of their phase separation capabilities. Moreover, modular domains (MDs), another class of multivalent molecules, though ineffective in enhancing dCas9-VP64 activity on their own, show substantial enhancement in transcriptional activation when combined with dCas9-VP64-IDR. By varying the number of gRNA binding sites and fusing dCas9-VP64 with different IDRs/MDs, we uncover that optimal, rather than maximal, cis-trans cooperativity enables the most robust activation. Finally, targeting promoter-enhancer pairs yields synergistic effects, which can be further amplified via enhancing chromatin interactions. Overall, our study develops a versatile platform for efficient gene activation and sheds important insights into CRIPSR-based transcriptional activators enhanced with multivalent molecules.

The Disorderly Nature of Caliciviruses.

An intrinsically disordered protein (IDP) or region (IDR) lacks or has little protein structure but still maintains function. This lack of structure creates flexibility and fluidity, allowing multiple protein conformations and potentially transient interactions with more than one partner. Caliciviruses are positive-sense ssRNA viruses, containing a relatively small genome of 7.6-8.6 kb and have a broad host range. Many viral proteins are known to contain IDRs, which benefit smaller viral genomes by expanding the functional proteome through the multifunctional nature of the IDR. The percentage of intrinsically disordered residues within the total proteome for each calicivirus type species can range between 8 and 23%, and IDRs have been experimentally identified in NS1-2, VPg and RdRP proteins. The IDRs within a protein are not well conserved across the genera, and whether this correlates to different activities or increased tolerance to mutations, driving virus adaptation to new selection pressures, is unknown. The function of norovirus NS1-2 has not yet been fully elucidated but includes involvement in host cell tropism, the promotion of viral spread and the suppression of host interferon-λ responses. These functions and the presence of host cell-like linear motifs that interact with host cell caspases and VAPA/B are all found or affected by the disordered region of norovirus NS1-2. The IDRs of calicivirus VPg are involved in viral transcription and translation, RNA binding, nucleotidylylation and cell cycle arrest, and the N-terminal IDR within the human norovirus RdRP could potentially drive liquid-liquid phase separation. This review identifies and summarises the IDRs of proteins within the Caliciviridae family and their importance during viral replication and subsequent host interactions.

Sequence-encoded Spatiotemporal Dependence of Viscoelasticity of Protein Condensates Using Computational Microrheology.

Many biomolecular condensates act as viscoelastic complex fluids with distinct cellular functions. Deciphering the viscoelastic behavior of biomolecular condensates can provide insights into their spatiotemporal organization and physiological roles within cells. Though there is significant interest in defining the role of condensate dynamics and rheology in physiological functions, the quantification of their time-dependent viscoelastic properties is limited and mostly done through experimental rheological methods. Here, we demonstrate that a computational passive probe microrheology technique, coupled with continuum mechanics, can accurately characterize the linear viscoelasticity of condensates formed by intrinsically disordered proteins (IDPs). Using a transferable coarse-grained protein model, we first provide a physical basis for choosing optimal values that define the attributes of the probe particle, namely its size and interaction strength with the residues in an IDP chain. We show that the technique captures the sequence-dependent viscoelasticity of heteropolymeric IDPs that differ either in sequence charge patterning or sequence hydrophobicity. We also illustrate the technique's potential in quantifying the spatial dependence of viscoelasticity in heterogeneous IDP condensates. The computational microrheology technique has important implications for investigating the time dependent rheology of complex biomolecular architectures, resulting in the sequence-rheology-function relationship for condensates.

Differential Effects of Sequence-Local versus Nonlocal Charge Patterns on Phase Separation and Conformational Dimensions of Polyampholytes as Model Intrinsically Disordered Proteins.

Conformational properties of intrinsically disordered proteins (IDPs) are governed by a sequence-ensemble relationship. To differentiate the impact of sequence-local versus sequence-nonlocal features of an IDP's charge pattern on its conformational dimensions and its phase-separation propensity, the charge "blockiness" κ and the nonlocality-weighted sequence charge decoration (SCD) parameters are compared for their correlations with isolated-chain radii of gyration (Rgs) and upper critical solution temperatures (UCSTs) of polyampholytes modeled by random phase approximation, field-theoretic simulation, and coarse-grained molecular dynamics. SCD is superior to κ in predicting Rg because SCD accounts for effects of contact order, i.e., nonlocality, on dimensions of isolated chains. In contrast, κ and SCD are comparably good, though nonideal, predictors of UCST because frequencies of interchain contacts in the multiple-chain condensed phase are less sensitive to sequence positions than frequencies of intrachain contacts of an isolated chain, as reflected by κ correlating better with condensed-phase interaction energy than SCD.