The Theranostic Optimization of PSMA-GCK01 Does Not Compromise the Imaging Characteristics of [99mTc]Tc-PSMA-GCK01 Compared to Dedicated Diagnostic [99mTc]Tc-EDDA/HYNIC-iPSMA in Prostate Cancer.

PURPOSE: Radiolabeled PSMA-ligands play a major role in today's nuclear medicine. Since approval of [177Lu]Lu-PSMA-617 for therapy of metastatic prostate cancer, availability of 177Lu became bottleneck of supply due to the high demand. Recently, a theranostic PSMA-ligand, PSMA-GCK01, was developed which can be labeled either diagnostically with 99mTc or therapeutically with 188Re with both nuclides available from well-known generator systems. This novel tracer might aid to overcome aforementioned supply limitations. In this investigation, the biodistribution and general imaging characteristics of [99mTc]Tc-PSMA-GCK01 were compared with the diagnostic reference compound [99mTc]Tc-EDDA/HYNIC-iPSMA in patients with advanced stage prostate cancer. In addition, the binding of both ligands to PSMA was analyzed at the molecular level using molecular docking. PROCEDURES: Two cohorts (n = 19 vs. n = 21) of patients with metastatic castration-resistant prostate cancer matched for age, tumor stage, and Gleason score underwent a planar gamma camera imaging with [99mTc]Tc-EDDA/HYNIC-iPSMA or [99mTc]Tc-PSMA-GCK01 prior to PSMA-ligand therapy for PSMA-phenotyping. The imaging data were retrospective analyzed for salivary gland, kidney, liver, soft tissue, and tumor uptake on a semi-automated ROI-analysis using HERMES Medical Solutions AB (HMS, Sweden). RESULTS: The data sets were semi-automated quantified on a ROI-based analysis. The tumor-to-background presented equal results of [99mTc]Tc-PSMA-GCK01 compared to [99mTc]Tc-EDDA/HYNIC-iPSMA. The physiological PSMA-positive organs like salivary gland presented also equal uptake in counts/MBq (salivary gland median 9.48 [99mTc]Tc-PSMA-GCK01 vs. median 9.11 [99mTc]Tc-EDDA/HYNIC-iPSMA), while liver-to-kidney ratio presented a slight shift to the liver parenchyma using [99mTc]Tc-PSMA-GCK01 (0.83) compared to [99mTc]Tc-EDDA/HYNIC-iPSMA (0.55) with no statistical significance. This is in agreement with the results from the docking study revealing only a minor difference in the docking scores for both ligands. CONCLUSIONS: The novel theranostic tracer [99mTc]Tc/[188Re]Re-PSMA-GCK01 demonstrates comparable general imaging characteristic with the reference compound [99mTc]Tc-EDDA/HYNIC-iPSMA. These results pave the way for the PSMA-targeting imaging and theranostic agents for a broader, rather low-cost, generator applied radio-ligand therapy utilization.

Effects of ion type and concentration on the structure and aggregation of the amyloid peptide A ß 16 - 22 $$ {\boldsymbol{\beta}}_{16-22} $$.

Among the various factors controlling the amyloid aggregation process, the influences of ions on the aggregation rate and the resulting structures are important aspects to consider, which can be studied by molecular simulations. There is a wide variety of protein force fields and ion models, raising the question of which model to use in such studies. To address this question, we perform molecular dynamics simulations of Aß16-22 , a fragment of the Alzheimer's amyloid ß peptide, using different protein force fields, AMBER99SB-disp (A99-d) and CHARMM36m (C36m), and different ion parameters. The influences of NaCl and CaCl2 at various concentrations are studied and compared with the systems without the addition of ions. Our results indicate a sensitivity of the peptide-ion interactions to the different ion models. In particular, we observe a strong binding of Ca2+ to residue E22 with C36m and also with the Åqvist ion model used together with A99-d, which slightly affects the monomeric Aß16-22 structures and the aggregation rate, but significantly affects the oligomer structures formed in the aggregation simulations. For example, at high Ca2+ concentrations, there was a switch from an antiparallel to a parallel ß-sheet. Such ionic influences are of biological relevance because local ion concentrations can change in vivo and could help explain the polymorphism of amyloid fibrils.

Integrative modeling of guanylate binding protein dimers.

Guanylate-binding proteins (GBPs) are essential interferon-γ-activated large GTPases that play a crucial role in host defense against intracellular bacteria and parasites. While their protective functions rely on protein polymerization, our understanding of the structural intricacies of these multimerized states remains limited. To bridge this knowledge gap, we present dimer models for human GBP1 (hGBP1) and murine GBP2 and 7 (mGBP2 and mGBP7) using an integrative approach, incorporating the crystal structure of hGBP1's GTPase domain dimer, crosslinking mass spectrometry, small-angle X-ray scattering, protein-protein docking, and molecular dynamics simulations. Our investigation begins by comparing the protein dynamics of hGBP1, mGBP2, and mGBP7. We observe that the M/E domain in all three proteins exhibits significant mobility and hinge motion, with mGBP7 displaying a slightly less pronounced motion but greater flexibility in its GTPase domain. These dynamic distinctions can be attributed to variations in the sequences of mGBP7 and hGBP1/mGBP2, resulting in different dimerization modes. Unlike hGBP1 and its close ortholog mGBP2, which exclusively dimerize through their GTPase domains, we find that mGBP7 exhibits three equally probable alternative dimer structures. The GTPase domain of mGBP7 is only partially involved in its dimerization, primarily due to an accumulation of negative charge, allowing mGBP7 to dimerize independently of GTP. Instead, mGBP7 exhibits a strong tendency to dimerize in an antiparallel arrangement across its stalks. The results of this work go beyond the sequence-structure-function relationship, as the sequence differences in mGBP7 and mGBP2/hGBP1 do not lead to different structures, but to different protein dynamics and dimerization. The distinct GBP dimer structures are expected to encode specific functions crucial for disrupting pathogen membranes.

A Hairpin Motif in the Amyloid-ß Peptide Is Important for Formation of Disease-Related Oligomers.

The amyloid-ß (Aß) peptide is associated with the development of Alzheimer's disease and is known to form highly neurotoxic prefibrillar oligomeric aggregates, which are difficult to study due to their transient, low-abundance, and heterogeneous nature. To obtain high-resolution information about oligomer structure and dynamics as well as relative populations of assembly states, we here employ a combination of native ion mobility mass spectrometry and molecular dynamics simulations. We find that the formation of Aß oligomers is dependent on the presence of a specific ß-hairpin motif in the peptide sequence. Oligomers initially grow spherically but start to form extended linear aggregates at oligomeric states larger than those of the tetramer. The population of the extended oligomers could be notably increased by introducing an intramolecular disulfide bond, which prearranges the peptide in the hairpin conformation, thereby promoting oligomeric structures but preventing conversion into mature fibrils. Conversely, truncating one of the ß-strand-forming segments of Aß decreased the hairpin propensity of the peptide and thus decreased the oligomer population, removed the formation of extended oligomers entirely, and decreased the aggregation propensity of the peptide. We thus propose that the observed extended oligomer state is related to the formation of an antiparallel sheet state, which then nucleates into the amyloid state. These studies provide increased mechanistic understanding of the earliest steps in Aß aggregation and suggest that inhibition of Aß folding into the hairpin conformation could be a viable strategy for reducing the amount of toxic oligomers.

Transition Networks Unveil Disorder-to-Order Transformations in Aß Caused by Glycosaminoglycans or Lipids.

The aggregation of amyloid-ß (Aß) peptides, particularly of Aß1-42, has been linked to the pathogenesis of Alzheimer's disease. In this study, we focus on the conformational change of Aß1-42 in the presence of glycosaminoglycans (GAGs) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids using molecular dynamics simulations. We analyze the conformational changes that occur in Aß by extracting the key structural features that are then used to generate transition networks. Using the same three features per network highlights the transitions from intrinsically disordered states ubiquitous in Aß1-42 in solution to more compact states arising from stable ß-hairpin formation when Aß1-42 is in the vicinity of a GAG molecule, and even more compact states characterized by a α-helix or ß-sheet structures when Aß1-42 interacts with a POPC lipid cluster. We show that the molecular mechanisms underlying these transitions from disorder to order are different for the Aß1-42/GAG and Aß1-42/POPC systems. While in the latter the hydrophobicity provided by the lipid tails facilitates the folding of Aß1-42, in the case of GAG there are hardly any intermolecular Aß1-42-GAG interactions. Instead, GAG removes sodium ions from the peptide, allowing stronger electrostatic interactions within the peptide that stabilize a ß-hairpin. Our results contribute to the growing knowledge of the role of GAGs and lipids in the conformational preferences of the Aß peptide, which in turn influences its aggregation into toxic oligomers and amyloid fibrils.

Comparative molecular dynamics simulations of pathogenic and non-pathogenic huntingtin protein monomers and dimers.

Polyglutamine expansion at the N-terminus of the huntingtin protein exon 1 (Htt-ex1) is closely associated with a number of neurodegenerative diseases, which result from the aggregation of the increased polyQ repeat. However, the underlying structures and aggregation mechanism are still poorly understood. We performed microsecond-long all-atom molecular dynamics simulations to study the folding and dimerization of Htt-ex1 (about 100 residues) with non-pathogenic and pathogenic polyQ lengths, and uncovered substantial differences. The non-pathogenic monomer adopts a long α-helix that includes most of the polyQ residues, which forms the interaction interface for dimerization, and a PPII-turn-PPII motif in the proline-rich region. In the pathogenic monomer, the polyQ region is disordered, leading to compact structures with many intra-protein interactions and the formation of short ß-sheets. Dimerization can proceed via different modes, where those involving the N-terminal headpiece bury more hydrophobic residues and are thus more stable. Moreover, in the pathogenic Htt-ex1 dimers the proline-rich region interacts with the polyQ region, which slows the formation of ß-sheets.

Discovery of All-d-Peptide Inhibitors of SARS-CoV-2 3C-like Protease.

During the replication process of SARS-CoV-2, the main protease of the virus [3-chymotrypsin-like protease (3CLpro)] plays a pivotal role and is essential for the life cycle of the pathogen. Numerous studies have been conducted so far, which have confirmed 3CLpro as an attractive drug target to combat COVID-19. We describe a novel and efficient next-generation sequencing (NGS) supported phage display selection strategy for the identification of a set of SARS-CoV-2 3CLpro targeting peptide ligands that inhibit the 3CL protease, in a competitive or noncompetitive mode, in the low µM range. From the most efficient l-peptides obtained from the phage display, we designed all-d-peptides based on the retro-inverso (ri) principle. They had IC50 values also in the low µM range and in combination, even in the sub-micromolar range. Additionally, the combination with Rutinprivir decreases 10-fold the IC50 value of the competitive inhibitor. The inhibition modes of these d-ri peptides were the same as their respective l-peptide versions. Our results demonstrate that retro-inverso obtained all-d-peptides interact with high affinity and inhibit the SARS-CoV-2 3CL protease, thus reinforcing their potential for further development toward therapeutic agents. The here described d-ri peptides address limitations associated with current l-peptide inhibitors and are promising lead compounds. Further optimization regarding pharmacokinetic properties will allow the development of even more potent d-peptides to be used for the prevention and treatment of COVID-19.

Domain motions, dimerization, and membrane interactions of the murine guanylate binding protein 2.

Guanylate-binding proteins (GBPs) are a group of GTPases that are induced by interferon-[Formula: see text] and are crucial components of cell-autonomous immunity against intracellular pathogens. Here, we examine murine GBP2 (mGBP2), which we have previously shown to be an essential effector protein for the control of Toxoplasma gondii replication, with its recruitment through the membrane of the parasitophorous vacuole and its involvement in the destruction of this membrane likely playing a role. The overall aim of our work is to provide a molecular-level understanding of the mutual influences of mGBP2 and the parasitophorous vacuole membrane. To this end, we performed lipid-binding assays which revealed that mGBP2 has a particular affinity for cardiolipin. This observation was confirmed by fluorescence microscopy using giant unilamellar vesicles of different lipid compositions. To obtain an understanding of the protein dynamics and how this is affected by GTP binding, mGBP2 dimerization, and membrane binding, assuming that each of these steps are relevant for the function of the protein, we carried out standard as well as replica exchange molecular dynamics simulations with an accumulated simulation time of more than 30 µs. The main findings from these simulations are that mGBP2 features a large-scale hinge motion in its M/E domain, which is present in each of the studied protein states. When bound to a cardiolipin-containing membrane, this hinge motion is particularly pronounced, leading to an up and down motion of the M/E domain on the membrane, which did not occur on a membrane without cardiolipin. Our prognosis is that this up and down motion has the potential to destroy the membrane following the formation of supramolecular mGBP2 complexes on the membrane surface.

SEC14-GOLD protein PATELLIN2 binds IRON-REGULATED TRANSPORTER1 linking root iron uptake to vitamin E.

Organisms require micronutrients, and Arabidopsis (Arabidopsis thaliana) IRON-REGULATED TRANSPORTER1 (IRT1) is essential for iron (Fe2+) acquisition into root cells. Uptake of reactive Fe2+ exposes cells to the risk of membrane lipid peroxidation. Surprisingly little is known about how this is avoided. IRT1 activity is controlled by an intracellular variable region (IRT1vr) that acts as a regulatory protein interaction platform. Here, we describe that IRT1vr interacted with peripheral plasma membrane SEC14-Golgi dynamics (SEC14-GOLD) protein PATELLIN2 (PATL2). SEC14 proteins bind lipophilic substrates and transport or present them at the membrane. To date, no direct roles have been attributed to SEC14 proteins in Fe import. PATL2 affected root Fe acquisition responses, interacted with ROS response proteins in roots, and alleviated root lipid peroxidation. PATL2 had high affinity in vitro for the major lipophilic antioxidant vitamin E compound α-tocopherol. Molecular dynamics simulations provided insight into energetic constraints and the orientation and stability of the PATL2-ligand interaction in atomic detail. Hence, this work highlights a compelling mechanism connecting vitamin E with root metal ion transport at the plasma membrane with the participation of an IRT1-interacting and α-tocopherol-binding SEC14 protein.

Structure and Dynamics of Human Chemokine CCL16-Implications for Biological Activity.

Human C-C motif ligand 16 (CCL16) is a chemokine that is distinguished by a large cleavable C-terminal extension of unknown significance. Conflicting data have been reported concerning its tissue distribution and modulation of expression, rendering the biological function of CCL16 enigmatic. Here, we report an integrated approach to the characterisation of this chemokine, including a re-assessment of its expression characteristics as well as a biophysical investigation with respect to its structure and dynamics. Our data indicate that CCL16 is chiefly synthesised by hepatocytes, without an appreciable response to mediators of inflammation, and circulates in the blood as a full-length protein. While the crystal structure of CCL16 confirms the presence of a canonical chemokine domain, molecular dynamics simulations support the view that the C-terminal extension impairs the accessibility of the glycosaminoglycan binding sites and may thus serve as an intrinsic modulator of biological activity.

Multiple Substrate Binding Mode-Guided Engineering of a Thermophilic PET Hydrolase.

Thermophilic polyester hydrolases (PES-H) have recently enabled biocatalytic recycling of the mass-produced synthetic polyester polyethylene terephthalate (PET), which has found widespread use in the packaging and textile industries. The growing demand for efficient PET hydrolases prompted us to solve high-resolution crystal structures of two metagenome-derived enzymes (PES-H1 and PES-H2) and notably also in complex with various PET substrate analogues. Structural analyses and computational modeling using molecular dynamics simulations provided an understanding of how product inhibition and multiple substrate binding modes influence key mechanistic steps of enzymatic PET hydrolysis. Key residues involved in substrate-binding and those identified previously as mutational hotspots in homologous enzymes were subjected to mutagenesis. At 72 °C, the L92F/Q94Y variant of PES-H1 exhibited 2.3-fold and 3.4-fold improved hydrolytic activity against amorphous PET films and pretreated real-world PET waste, respectively. The R204C/S250C variant of PES-H1 had a 6.4 °C higher melting temperature than the wild-type enzyme but retained similar hydrolytic activity. Under optimal reaction conditions, the L92F/Q94Y variant of PES-H1 hydrolyzed low-crystallinity PET materials 2.2-fold more efficiently than LCC ICCG, which was previously the most active PET hydrolase reported in the literature. This property makes the L92F/Q94Y variant of PES-H1 a good candidate for future applications in industrial plastic recycling processes.

Does the inclusion of electronic polarisability lead to a better modelling of peptide aggregation?

Simulating the process of amyloid aggregation with atomic detail is a challenging task for various reasons. One of them is that it is difficult to parametrise a force field such that all protein states ranging from the folded through the unfolded to the aggregated state are represented with the same level of accuracy. Here, we test whether the consideration of electronic polarisability improves the description of the different states of Aß16-22. Surprisingly, the CHARMM Drude polarisable force field is found to perform worse than its unpolarisable counterpart CHARMM36m. Sources for this failure of the Drude model are discussed.

ATRANET - Automated generation of transition networks for the structural characterization of intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) do not fold into a unique three-dimensional structure but sample different configurations of different probabilities that further change with the surrounding of the IDPs. The structural heterogeneity and dynamics of IDPs pose a challenge for the characterization of their structures by experimental techniques only. Molecular dynamics (MD) simulations provide a powerful complement to experimental approaches for that purpose. However, MD simulations on the micro- to millisecond timescale generate a lot of data of protein motions, necessitating advanced post-processing techniques to extract the relevant information. Here, we demonstrate how transition networks created from MD trajectories allow revealing the configurational ensemble and structural interconversions of IDPs, using the amyloid-ß peptide as example. The construction of transition networks relies on molecular descriptors as input, and we show how the choice of descriptors influences the resulting transition network. The transition networks are generated with the open-source Python script ATRANET, and we explain the usage of ATRANET by providing a detailed workflow and exemplary analysis for amyloid-ß, which can be easily generalized to other IDPs and even protein aggregation.

Structural Dissection of the First Events Following Membrane Binding of the Islet Amyloid Polypeptide.

The islet amyloid polypeptide (IAPP) is the main constituent of the amyloid fibrils found in the pancreas of type 2 diabetes patients. The aggregation of IAPP is known to cause cell death, where the cell membrane plays a dual role: being a catalyst of IAPP aggregation and being the target of IAPP toxicity. Using ATR-FTIR spectroscopy, transmission electron microscopy, and molecular dynamics simulations we investigate the very first molecular steps following IAPP binding to a lipid membrane. In particular, we assess the combined effects of the charge state of amino-acid residue 18 and the IAPP-membrane interactions on the structures of monomeric and aggregated IAPP. Distinct IAPP-membrane interaction modes for the various IAPP variants are revealed. Membrane binding causes IAPP to fold into an amphipathic α-helix, which in the case of H18K-, and H18R-IAPP readily moves beyond the headgroup region. For all IAPP variants but H18E-IAPP, the membrane-bound helix is an intermediate on the way to amyloid aggregation, while H18E-IAPP remains in a stable helical conformation. The fibrillar aggregates of wild-type IAPP and H18K-IAPP are dominated by an antiparallel ß-sheet conformation, while H18R- and H18A-IAPP exhibit both antiparallel and parallel ß-sheets as well as amorphous aggregates. Our results emphasize the decisive role of residue 18 for the structure and membrane interaction of IAPP. This residue is thus a good therapeutic target for destabilizing membrane-bound IAPP fibrils to inhibit their toxic actions.

Multiscale MD simulations of wild-type and sickle hemoglobin aggregation.

Sickle cell disease is a hemoglobinopathy resulting from a point mutation from glutamate to valine at position six of the ß-globin chains of hemoglobin. This mutation gives rise to pathological aggregation of the sickle hemoglobin and, as a result, impaired oxygen binding, misshapen and short-lived erythrocytes, and anemia. We aim to understand the structural effects caused by the single Glu6Val mutation leading to protein aggregation. To this end, we perform multiscale molecular dynamics simulations employing atomistic and coarse-grained models of both wild-type and sickle hemoglobin. We analyze the dynamics of hemoglobin monomers and dimers, study the aggregation of wild-type and sickle hemoglobin into decamers, and analyze the protein-protein interactions in the resulting aggregates. We find that the aggregation of sickle hemoglobin is driven by both hydrophobic and electrostatic protein-protein interactions involving the mutation site and surrounding residues, leading to an extended interaction area and thus stable aggregates. The wild-type protein can also self-assemble, which, however, results from isolated interprotein salt bridges that do not yield stable aggregates. This knowledge can be exploited for the development of sickle hemoglobin-aggregation inhibitors.

Molecular Dynamics Simulations of Protein Aggregation: Protocols for Simulation Setup and Analysis with Markov State Models and Transition Networks.

Protein disorder and aggregation play significant roles in the pathogenesis of numerous neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. The end products of the aggregation process in these diseases are highly structured amyloid fibrils. Though in most cases, small, soluble oligomers formed during amyloid aggregation are the toxic species. A full understanding of the physicochemical forces that drive protein aggregation is thus required if one aims for the rational design of drugs targeting the formation of amyloid oligomers. Among a multitude of biophysical and biochemical techniques that are employed for studying protein aggregation, molecular dynamics (MD) simulations at the atomic level provide the highest temporal and spatial resolution of this process, capturing key steps during the formation of amyloid oligomers. Here we provide a step-by-step guide for setting up, running, and analyzing MD simulations of aggregating peptides using GROMACS. For the analysis, we provide the scripts that were developed in our lab, which allow to determine the oligomer size and inter-peptide contacts that drive the aggregation process. Moreover, we explain and provide the tools to derive Markov state models and transition networks from MD data of peptide aggregation.

Disorder-to-order transition of the amyloid-ß peptide upon lipid binding.

There is mounting evidence that Alzheimer's disease progression and severity are linked to neuronal membrane damage caused by aggregates of the amyloid-ß (Aß) peptide. However, the detailed mechanism behind the membrane damage is not well understood yet. Recently, the lipid-chaperone hypothesis has been put forward, based on which the formation of complexes between Aß and free lipids enables an easy insertion of Aß into membranes. In order to test this hypothesis, we performed numerous all-atom molecular dynamics simulations. We studied the complex formation between individual lipids, considering both POPC and DPPC, and Aß and examined whether the resulting complexes would be able to insert into lipid membranes. Complex formation at a one-to-one ratio was readily observed, yet with minimal effects on Aß's characteristics. Most importantly, the peptide remains largely disordered in 1:1 complexes, and the complex does not insert into the membrane; instead, it is adsorbed to the membrane surface. The results change considerably once Aß forms a complex with a POPC cluster composed of three lipid molecules. The hydrophobic interactions between Aß and the lipid tails cause the peptide to fold into either a helical or a ß-sheet structure. These observations provide atomic insight into the disorder-to-order transition that is needed for membrane insertion or amyloid aggregation to proceed.

The Influences of Sulphation, Salt Type, and Salt Concentration on the Structural Heterogeneity of Glycosaminoglycans.

The increasing recognition of the biochemical importance of glycosaminoglycans (GAGs) has in recent times made them the center of attention of recent research investigations. It became evident that subtle conformational factors play an important role in determining the relationship between the chemical composition of GAGs and their activity. Therefore, a thorough understanding of their structural flexibility is needed, which is addressed in this work by means of all-atom molecular dynamics (MD) simulations. Four major GAGs with different substitution patterns, namely hyaluronic acid as unsulphated GAG, heparan-6-sulphate, chondroitin-4-sulphate, and chondroitin-6-sulphate, were investigated to elucidate the influence of sulphation on the dynamical features of GAGs. Moreover, the effects of increasing NaCl and KCl concentrations were studied as well. Different structural parameters were determined from the MD simulations, in combination with a presentation of the free energy landscape of the GAG conformations, which allowed us to unravel the conformational fingerprints unique to each GAG. The largest effects on the GAG structures were found for sulphation at position 6, as well as binding of the metal ions in the absence of chloride ions to the carboxylate and sulphate groups, which both increase the GAG conformational flexibility.

Molecular simulations of IDPs: From ensemble generation to IDP interactions leading to disorder-to-order transitions.

Intrinsically disordered proteins (IDPs) lack a well-defined three-dimensional structure but do exhibit some dynamical and structural ordering. The structural plasticity of IDPs indicates that entropy-driven motions are crucial for their function. Many IDPs undergo function-related disorder-to-order transitions upon by their interaction with specific binding partners. Approaches that are based on both experimental and theoretical tools enable the biophysical characterization of IDPs. Molecular simulations provide insights into IDP structural ensembles and disorder-to-order transition mechanisms. However, such studies depend strongly on the chosen force field parameters and simulation techniques. In this chapter, we provide an overview of IDP characteristics, review all-atom force fields recently developed for IDPs, and present molecular dynamics-based simulation methods that allow IDP ensemble generation as well as the characterization of disorder-to-order transitions. In particular, we introduce metadynamics, replica exchange molecular dynamics simulations, and also kinetic models resulting from Markov State modeling, and provide various examples for the successful application of these simulation methods to IDPs.