Distinct motifs in the E protein are required for SARS-CoV-2 virus particle formation and lysosomal deacidification in host cells.

IMPORTANCE: Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19), has caused a global public health crisis. The E protein, a structural protein found in this virus particle, is also known to be a viroporin. As such, it forms oligomeric ion channels or pores in the host cell membrane. However, the relationship between these two functions is poorly understood. In this study, we showed that the roles of E protein in virus particle and viroporin formation are distinct. This study contributes to the development of drugs that inhibit SARS-CoV-2 virus particle formation. Additionally, we designed a highly sensitive and high-throughput virus-like particle detection system using the HiBiT tag, which is a useful tool for studying the release of SARS-CoV-2.

Molecular mechanisms of amyloid-ß peptide fibril and oligomer formation: NMR-based challenges.

To completely treat and ultimately prevent dementia, it is essential to elucidate its pathogenic mechanisms in detail. There are two major hypotheses for the pathogenesis of Alzheimer's dementia: the ß-amyloid (Aß) hypothesis and the tau hypothesis. The modified amyloid hypothesis, which proposes that toxic oligomers rather than amyloid fibrils are the essential cause, has recently emerged. Aß peptides [Aß(1-40) and Aß(1-42)] form highly insoluble aggregates in vivo and in vitro. These Aß aggregates contain many polymorphisms, whereas Aß peptides are intrinsically disordered in physiological aqueous solutions without any compact conformers. Over the last three decades, solid-state nuclear magnetic resonance (NMR) has greatly contributed to elucidating the structure of each polymorph, while solution NMR has revealed the dynamic nature of the transient conformations of the monomer. Moreover, several methods to investigate the aggregation process based on the observation of magnetization saturation transfer have also been developed. The complementary use of NMR methods with cryo-electron microscopy, which has rapidly matured, is expected to clarify the relationship between the amyloid and molecular pathology of Alzheimer's dementia in the near future. This review article is an extended version of the Japanese article, Insights into the Mechanisms of Oligomerization/Fibrilization of Amyloid ß Peptide from Nuclear Magnetic Resonance, published in SEIBUTSU BUTSURI Vol. 62, p. 39-42 (2022).

A Cost-Effective Immobilization Method for MBP Fusion Proteins on Microtiter Plates Using a Gelatinized Starch-Agarose Mixture and Its Application for Convenient Protein-Protein Interaction Analysis.

The detection and quantification of protein-protein interactions (PPIs) is a crucial technique that often involves the use of recombinant proteins with fusion protein tags, such as maltose-binding protein (MBP) and glutathione-S-transferase (GST). In this study, we improved the cohesive and sticky properties of gelatinized starch by supplementing it with agarose, resulting in a harder gel that could coat the bottom of a microtiter plate. The resulting gelatinized starch/agarose mixture allowed for the efficient immobilization of MBP-tagged proteins on the coated plates, enabling the use of indirect ELISA-like PPI assays. By using the enzymatic activity of GST as an indicator, we succeeded in determining the dissociation constants between MBP-tagged and GST-tagged proteins on 96-well microtiter plates and a microplate reader without any expensive specialized equipment.

Pharmacologic Comparison of High-Dose Hesperetin and Quercetin on MDCK II Cell Viability, Tight Junction Integrity, and Cell Shape.

The modulation of tight junction (TJ) integrity with small molecules is important for drug delivery. High-dose baicalin (BLI), baicalein (BLE), quercetin (QUE), and hesperetin (HST) have been shown to open TJs in Madin-Darby canine kidney (MDCK) II cells, but the mechanisms for HST and QUE remain unclear. In this study, we compared the effects of HST and QUE on cell proliferation, morphological changes, and TJ integrity. HST and QUE were found to have opposing effects on the MDCK II cell viability, promotion, and suppression, respectively. Only QUE, but not HST, induced a morphological change in MDCK II into a slenderer cell shape. Both HST and QUE downregulated the subcellular localization of claudin (CLD)-2. However, only QUE, but not HST, downregulated CLD-2 expression. Conversely, only HST was shown to directly bind to the first PDZ domain of ZO-1, a key molecule to promote TJ biogenesis. The TGFß pathway partially contributed to the HST-induced cell proliferation, since SB431541 ameliorated the effect. In contrast, the MEK pathway was not involved by both the flavonoids, since U0126 did not revert their TJ-opening effect. The results offer insight for using HST or QUE as naturally occurring absorption enhancers through the paracellular route.

Small-Molecule-Mediated Suppression of BMP Signaling by Selective Inhibition of BMP1-Dependent Chordin Cleavage.

BMP signaling is critical for many biological processes. Therefore, small molecules that modulate BMP signaling are useful for elucidating the function of BMP signaling and treating BMP signaling-related diseases. Here, we performed a phenotypic screening in zebrafish to examine the in vivo effects of N-substituted-2-amino-benzoic acid analogs NPL1010 and NPL3008 and found that they affect BMP signaling-dependent dorsal-ventral (D-V) patterning and bone formation in zebrafish embryos. Furthermore, NPL1010 and NPL3008 suppressed BMP signaling upstream of BMP receptors. BMP1 cleaves Chordin, an antagonist of BMP, and negatively regulates BMP signaling. Docking simulations demonstrated that NPL1010 and NPL3008 bind BMP1. We found that NPL1010 and NPL3008 partially rescued the disruptions in the D-V phenotype caused by bmp1 overexpression and selectively inhibited BMP1-dependent Chordin cleavage. Therefore, NPL1010 and NPL3008 are potentially valuable inhibitors of BMP signaling that act through selective inhibition of Chordin cleavage.

A cryptic phosphate-binding pocket on the SPFH domain of human stomatin that regulates a novel fibril-like self-assembly.

Human stomatin (hSTOM) is a component of the membrane skeleton of erythrocytes that maintains the membrane's shape and stiffness through interconnecting spectrin and actin. hSTOM is a member of the protein family that possesses a single stomatin/prohibitin/flotillin/HflK (SPFH) domain at the center of the molecule. Although SPFH domain proteins are widely distributed from archaea to mammals, the detailed function of the domain remains unclear. In this study, we first determined the solution structure of the SPFH domain of hSTOM (hSTOM(SPFH)) via NMR. The solution structure of hSTOM(SPFH) is essentially identical to the already reported crystal structure of the STOM SPFH domain (mSTOM(SPFH)) of mice, except for the existence of a small hydrophilic pocket on the surface. We identified this pocket as a phosphate-binding site by comparing its NMR spectra with and without phosphate ions. Meanwhile, during the conventional process of protein NMR analysis, we eventually discovered that hSTOM(SPFH) formed a unique solid material after lyophilization. This lyophilized hSTOM(SPFH) sample was moderately slowly dissolved in a physiological buffer. Interestingly, it was resistant to dissolution against the phosphate buffer. We then found that the lyophilized hSTOM(SPFH) formed a fibril-like assembly under electron microscopy. Finally, we succeeded in reproducing this fibril-like assembly of hSTOM(SPFH) using a centrifugal ultrafiltration device, thus demonstrating that the increased protein concentration may promote self-assembly of hSTOM(SPFH) into fibril forms. Our observations may help understand the molecular function of the SPFH domain and its involvement in protein oligomerization as a component of the membrane skeleton. (245 words).

Relevance of Amorphous and Amyloid-Like Aggregates of the p53 Core Domain to Loss of its DNA-Binding Activity.

The anti-oncogenic protein p53 is a transcription factor that prevents tumorigenesis by inducing gene repair proteins or apoptosis under DNA damage. Since the DNA-binding domain of p53 (p53C) is aggregation-prone, the anti-oncogenic function of p53 is often lost in cancer cells. This tendency is rather severe in some tumor-related p53 mutants, such as R175H. In this study, we examined the effect of salts, including KCl and sugars, on the aggregation of p53C by monitoring two distinct aggregates: amorphous-like and amyloid-like. The amorphous aggregates are detectable with 8-(phenylamino)-1-naphthalenesulfonic acid (ANS) fluorescence, whereas the amyloid aggregates are sensitive to thioflavin-T (ThT) fluorescence. We found that KCl inhibited the formation of amorphous aggregates but promoted the formation of amyloid aggregates in a p53C R175H mutant. The salts exhibited different effects against the wild-type and R175H mutants of p53C. However, the ratio of ANS/ThT fluorescence for the wild-type and R175H mutant remained constant. KCl also suppressed the structural transition and loss of the DNA-binding function of p53C. These observations indicate the existence of multiple steps of p53C aggregation, probably coupled with the dissociation of Zn. Notably, amorphous aggregates and amyloid aggregates have distinct properties that could be discriminated by various small additives upon aggregation.

Potential of rescue and reactivation of tumor suppressor p53 for cancer therapy.

The tumor suppressor protein p53, a transcription product of the anti-oncogene TP53, is a critical factor in preventing cellular cancerization and killing cancer cells by inducing apoptosis. As a result, p53 is often referred to as the "guardian of the genome." Almost half of cancers possess genetic mutations in the TP53 gene, and most of these mutations result in the malfunction of p53, which promotes aggregation. In some cases, the product of the TP53 mutant allele shows higher aggregation propensity; the mutant co-aggregates with the normal (functional) p53 protein, thus losing cellular activity of the p53 guardian. Cancer might also progress because of the proteolytic degradation of p53 by activated E3 ubiquitination enzymes, MDM2 and MDM4. The inhibition of the specific interaction between MDM2 (MDM4) and p53 also results in increased p53 activity in cancer cells. Although the molecular targets of the drugs are different, two drug discovery strategies with a common goal, "rescuing p53 protein," have recently emerged. To conduct this approach, various biophysical methods of protein characterization were employed. In this review, we focus on these two independent strategies based on the unique biophysical features of the p53 protein.

Direct inhibition of the first PDZ domain of ZO-1 by glycyrrhizin is a possible mechanism of tight junction opening of Caco-2 cells.

Glycyrrhizin (GL) is known to exhibit a variety of useful pharmacological activities, including anti-inflammation, anti-hepatotoxicity, and enhancement of intestinal drug absorption. GL has been reported to modify the assembly of actin filaments, thereby modulating tight junction (TJ) integrity, but the detailed molecular mechanisms of this remain unclear. In this study, we first found that GL binds to the first PDZ domain of zonula occludens-1 (ZO-1(PDZ1)) through NMR experiments. The structure of the GL-ZO-1(PDZ1) complex was then constructed using HADDOCK with the transferred nuclear Overhauser effect-based inter-hydrogen distance constraints as well as restrictions on the interfacial residues identified from 1H-15N HSQC spectral changes. We identified the relevant interactions between the glucuronate-2 moiety of GL and the carboxylate binding loop of the ligand binding site of ZO-1(PDZ1). We further examined the interaction of ZO-1(PDZ1) with glycyrrhetinic acid and with GA-3-monoglucuronide and observed a much lower affinity for each than for that with GL, with good agreement with the model. The other contacts found in the model were examined by using an amino acid substitution mutant of ZO-1(PDZ1). Finally, we reproduced the experiments reported by Sakai et al. in which high-dose GL prolonged the TJ-opening mediated with sodium deoxycholate as indicated by reduced transepithelial electrical resistance.

NMR-Guided Repositioning of Non-Steroidal Anti-Inflammatory Drugs into Tight Junction Modulators.

Bioavailability is a major bottleneck in the clinical application of medium molecular weight therapeutics, including protein and peptide drugs. Paracellular transport of these molecules is hampered by intercellular tight junction (TJ) complexes. Therefore, safe chemical regulators for TJ loosening are desired. Here, we showed a potential application of select non-steroidal anti-inflammatory drugs (NSAIDs) as TJ modulators. Based on our previous observation that diclofenac and flufenamic acid directly bound various PDZ domains with a broad specificity, we applied solution nuclear magnetic resonance techniques to examine the interaction of other NSAIDs and the first PDZ domain (PDZ1) of zonula occludens (ZO)-1, ZO-1(PDZ1). Inhibition of ZO-1(PDZ1) is expected to provide loosening of the epithelial barrier function because the domain plays a crucial role in maintaining TJ integrity. Accordingly, diclofenac and indomethacin were found to decrease the subcellular localization of claudin (CLD)-2 but not occludin and ZO-1 at the apicolateral intercellular compartment of Madin-Darby canine kidney (MDCK) II cells. These NSAIDs exhibited 125-155% improved paracellular efflux of fluorescein isothiocyanate insulin for the Caco-2 cell monolayer. We propose that these NSAIDs can be repurposed as drug absorption enhancers for peptide drugs.

Opposing Effect of Naringenin and Quercetin on the Junctional Compartment of MDCK II Cells to Modulate the Tight Junction.

Maintaining tight junction (TJ) integrity is important for epithelial cell barriers. Previously, the enhancement of TJ integrity, induced by citrus-derived flavonoids, naringin (NRG) and hesperidin (HSD), was demonstrated, but the effects of their aglycones naringenin (NAR) and hesperetin (HST), and the mechanisms, have not been systematically investigated. Here we compared three series of flavonoids related to NAR, HST, quercetin (QUE) and their glycosides with the Madin-Darby canine kidney (MDCK) II cell monolayers. The effect of flavonoids on the protein expression level of claudin (CLD)-2 and its subcellular localization were investigated. NAR, NRG, and HSD increased the CLD-2 localization at the TJ compartment, and its protein expression level. QUE and HST showed TJ-mitigating activity. Narirutin (NRT), neohesperidin (NHD) and rutin (RUT) did not affect the TJ. In addition, NAR and QUE induced an increase or decrease of the transepithelial electrical resistance (TEER) values of the MDCK II monolayers. Two known signaling pathways, phosphatidyl-inositol-3 kinase (PI3K) and 5'-AMP-activated protein kinase (AMPK), were further compared with NAR. Two-dimensional polyacrylamide electrophoresis (2D PAGE) analysis of whole-cell proteins treated with NAR, AICA-riboside (AMPK activator) and LY294002 (PI3K inhibitor) showed in both a distinct pattern. This suggests the target of NAR's CLD-2 or zonula occludens-1 (ZO-1) modulation was unique.

High dose of baicalin or baicalein can reduce tight junction integrity by partly targeting the first PDZ domain of zonula occludens-1 (ZO-1).

The tight junction (TJ) is the apical-most intercellular junction complex, serving as a biological barrier of intercellular spaces between epithelial cells. The TJ's integrity is maintained by a key protein-protein interaction between C-terminal motifs of claudins (CLDs) and the postsynaptic density 95 (PSD-95)/discs large/zonula occludens 1 (ZO-1; PDZ) domains of ZO-1. Weak but direct interaction of baicalin and its aglycon, baicalein-which are pharmacologically active components of Chinese skullcap (Radix scutellariae)-with ZO-1(PDZ1) have been observed in NMR experiments. Next, we observed TJ-mitigating activity of these flavonoids against Madin-Darby canine kidney (MDCK) II cells with the downregulation of subcellular localization of CLD-2 at TJs. Meanwhile, baicalein-but not baicalin-induced a slender morphological change of MDCK cells' shape from their normal cobblestone-like shapes. Since baicalin and baicalein did not induce a localization change of occludin (OCLN), a "partial" epithelial-mesenchymal transition (EMT) induced by these flavonoids was considered. SB431542, an ALK-5 inhibitor, reversed the CLD-2 downregulation of both baicalin and baicalein, while SB431542 did not reverse the slender morphology. In contrast, the MEK/ERK inhibitor U0126 reversed the slender shape change. Thus, in addition to inhibition of the ZO-1-CLD interaction, activation of both transforming growth factor-ß (TGF-ß) and MEK/ERK signaling pathways have been suggested to be involved in TJ reduction by these flavonoids. Finally, we demonstrated that baicalin enhanced the permeability of fluorescence-labeled insulin via the paracellular pathway of the Caco-2 cell layer. We propose that baicalin, baicalein, and Radix scutellariae extract are useful as drug absorption enhancers.

Presence of intrinsically disordered proteins can inhibit the nucleation phase of amyloid fibril formation of Aß(1-42) in amino acid sequence independent manner.

The molecular shield effect was studied for intrinsically disordered proteins (IDPs) that do not adopt compact and stable protein folds. IDPs are found among many stress-responsive gene products and cryoprotective- and drought-protective proteins. We recently reported that some fragments of human genome-derived IDPs are cryoprotective for cellular enzymes, despite a lack of relevant amino acid sequence motifs. This sequence-independent IDP function may reflect their molecular shield effect. This study examined the inhibitory activity of IDPs against fibril formation in an amyloid beta peptide (Aß(1-42)) model system. Four of five human genome-derived IDPs (size range 20 to 44 amino acids) showed concentration-dependent inhibition of amyloid formation (IC50 range between 60 and 130 µM against 20 µM Aß(1-42)). The IC50 value was two orders of magnitude lower than that of polyethylene-glycol and dextran, used as neutral hydrophilic polymer controls. Nuclear magnetic resonance with 15 N-labeled Aß(1-42) revealed no relevant molecular interactions between Aß(1-42) and IDPs. The inhibitory activities were abolished by adding external amyloid-formation seeds. Therefore, IDPs seemed to act only at the amyloid nucleation phase but not at the elongation phase. These results suggest that IDPs (0.1 mM or less) have a molecular shield effect that prevents aggregation of susceptible molecules.

Principal component analysis of data from NMR titration experiment of uniformly 15N labeled amyloid beta (1-42) peptide with osmolytes and phenolic compounds.

A simple NMR method to analyze the data obtained by NMR titration experiment of amyloid formation inhibitors against uniformly 15N-labeled amyloid-ß 1-42 peptide (Aß(1-42)) was described. By using solution nuclear magnetic resonance (NMR) measurement, the simplest method for monitoring the effects of Aß fibrilization inhibitors is the NMR chemical shift perturbation (CSP) experiment using 15N-labeled Aß(1-42). However, the flexible and dynamic nature of Aß(1-42) monomer may hamper the interpretation of CSP data. Here we introduced principal component analysis (PCA) for visualizing and analyzing NMR data of Aß(1-42) in the presence of amyloid inhibitors including high concentration osmolytes. We measured 1H-15N 2D spectra of Aß(1-42) at various temperatures as well as of Aß(1-42) with several inhibitors, and subjected all the data to PCA (PCA-HSQC). The PCA diagram succeeded in differentiating the various amyloid inhibitors, including epigallocatechin gallate (EGCg), rosmarinic acid (RA) and curcumin (CUR) from high concentration osmolytes. We hypothesized that the CSPs reflected the conformational equilibrium of intrinsically disordered Aß(1-42) induced by weak inhibitor binding rather than the specific molecular interactions.

Synthesis of selenomethylene-locked nucleic acids (SeLNA) nucleoside unit bearing an adenine base.

Synthesis of selenomethylene-locked nucleic acids nucleoside bearing an adenine base (SeLNA-A) was investigated. We first examined the stereoinversion reaction at 2'-positions of a 5',3'-O-TIPDS-protected 4'-C-(hydroxymethyl)ribosyladenine derivative to give the corresponding arabinosyladenine. After triflation, treatment of the arabinosyladenine derivative with a mixture of selenium and sodium borohydride in ethanol managed to construct the desired SeLNA skeleton. Finally, removal of TIPDS by treating with fluoride gave the SeLNA-A nucleoside. In this study, we found the heat-labile property of SeLNA-A. It is necessary to know more precise characteristics of SeLNA to achieve its oligonucleotides synthesis.

Discovery of Potent Disheveled/Dvl Inhibitors Using Virtual Screening Optimized With NMR-Based Docking Performance Index.

Most solid tumors have their own cancer stem cells (CSCs), which are resistant to standard chemo-therapies. Recent reports have described that Wnt pathway plays a key role in self-renewal and tumorigenesis of CSCs. Regarding the Wnt/ß-catenin pathway, Dvl (mammalian Disheveled) is an attractive target of drug discovery. After analyzing the PDZ domain of human Dvl1 (Dvl1-PDZ) using NMR, we subjected it to preliminary NMR titration studies with 17 potential PDZ-binding molecules including CalBioChem-322338, a commercially available Dvl PDZ domain inhibitor. Next, we performed virtual screening (VS) using the program GOLD with nine parameter sets. Results were evaluated using the NMR-derived docking performance index (NMR-DPI). One parameter set of GOLD docking showing the best NMR-DPI was selected and used for the second VS against 5,135 compounds. The second docking trial identified more than 1,700 compounds that exhibited higher scores than CalBioChem-322338. Subsequent NMR titration experiments with five new candidate molecules (NPL-4001, 4004, 4011, 4012, and 4013), Dvl1-PDZ revealed larger chemical shift changes than those of CalBioChem-322338. Finally, these compounds showed partial proliferation inhibition activity against BT-20, a triple negative breast cancer (TNBC) cell. These compounds are promising Wnt pathway inhibitors that are potentially useful for anti-TNBC therapy.

Spatial Overlap of Claudin- and Phosphatidylinositol Phosphate-Binding Sites on the First PDZ Domain of Zonula Occludens 1 Studied by NMR.

Background: The tight junction is an intercellular adhesion complex composed of claudins (CLDs), occludin, and the scaffolding proteins zonula occludens 1 (ZO-1) and its two paralogs ZO-2 and ZO-3. ZO-1 is a multifunctional protein that contains three PSD95/Discs large/ZO-1(PDZ) domains. A key functional domain of ZO-1 is the first PDZ domain (ZO-1(PDZ1)) that recognizes the conserved C-termini of CLDs. Methods: In this study, we confirmed that phosphoinositides bound directly to ZO-1(PDZ1) by biochemical and solution NMR experiments. We further determined the solution structure of mouse ZO-1(PDZ1) by NMR and mapped the phosphoinositide binding site onto its molecular surface. Results: The phosphoinositide binding site was spatially overlapped with the CLD-binding site of ZO-1(PDZ1). Accordingly, inositol-hexaphosphate (phytic acid), an analog of the phosphoinositide head group, competed with ZO-1(PDZ)-CLD interaction. Conclusions: The results suggested that the PDZ domain⁻phosphoinositide interaction plays a regulatory role in biogenesis and homeostasis of the tight junction.

Using 1 HN amide temperature coefficients to define intrinsically disordered regions: An alternative NMR method.

This report describes a cost-effective experimental method for determining an intrinsically disordered protein (IDP) region in a given protein sample. In this area, the most popular (and conventional) means is using the amide (1 HN ) NMR signal chemical shift distributed in the range of 7.5-8.5 ppm. For this study, we applied an additional step: analysis of 1 HN chemical shift temperature coefficients (1 HN -CSTCs) of the signals. We measured 1 H-15 N two-dimensional NMR spectra of model IDP samples and ordered samples at four temperatures (288, 293, 298, and 303 K). We derived the 1 HN -CSTC threshold deviation, which gives the best correlation of ordered and disordered regions among the proteins examined (below -3.6 ppb/K). By combining these criteria with the newly optimized chemical shift range (7.8-8.5 ppm), the ratios of both true positive and true negative were improved by approximately 19% (62-81%) compared with the conventional "chemical shift-only" method.

Discovery of Cryoprotective Activity in Human Genome-Derived Intrinsically Disordered Proteins.

Intrinsically disordered proteins (IDPs) are an emerging phenomenon. They may have a high degree of flexibility in their polypeptide chains, which lack a stable 3D structure. Although several biological functions of IDPs have been proposed, their general function is not known. The only finding related to their function is the genetically conserved YSK2 motif present in plant dehydrins. These proteins were shown to be IDPs with the YSK2 motif serving as a core region for the dehydrins' cryoprotective activity. Here we examined the cryoprotective activity of randomly selected IDPs toward the model enzyme lactate dehydrogenase (LDH). All five IDPs that were examined were in the range of 35-45 amino acid residues in length and were equally potent at a concentration of 50 µg/mL, whereas folded proteins, the PSD-95/Dlg/ZO-1 (PDZ) domain, and lysozymes had no potency. We further examined their cryoprotective activity toward glutathione S-transferase as an example of the other enzyme, and toward enhanced green fluorescent protein as a non-enzyme protein example. We further examined the lyophilization protective activity of the peptides toward LDH, which revealed that some IDPs showed a higher activity than that of bovine serum albumin (BSA). Based on these observations, we propose that cryoprotection is a general feature of IDPs. Our findings may become a clue to various industrial applications of IDPs in the future.

Common molecular pathogenesis of disease-related intrinsically disordered proteins revealed by NMR analysis.

Intrinsically disordered proteins (IDPs) are either completely unstructured or contain large disordered regions in their native state; they have drawn much attention in the field of molecular pathology. Some of them substantially tend to form protein self-assemblies, such as toxic or non-toxic aggregates and fibrils, and have been postulated to relate to diseases. These disease-related IDPs include Aß(1-42) [Alzheimer's disease (AD)], Tau (AD and tauopathy), α-synuclein (Parkinson's disease) and p53 (cancer). Several studies suggest that these aggregation and/or fibril formation processes are often initiated by transient conformational changes of the IDPs prior to protein self-assembly. Interestingly, the pathological molecular processes of these IDPs share multiple common features with those of protein misfolding diseases, such as transmissible spongiform encephalopathy (PrPsc) and AL-amyloidosis (VL-domain of γ-immunoglobulin). This review provides an overview of solution NMR techniques that can help analyse the early and transient events of conformational equilibrium of IDPs and folded proteins.