Optimal 13C NMR investigation of intrinsically disordered proteins at 1.2 GHz.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for characterizing biomolecules such as proteins and nucleic acids at atomic resolution. Increased magnetic field strengths drive progress in biomolecular NMR applications, leading to improved performance, e.g., higher resolution. A new class of NMR spectrometers with a 28.2 T magnetic field (1.2 GHz 1H frequency) has been commercially available since the end of 2019. The availability of ultra-high-field NMR instrumentation makes it possible to investigate more complex systems using NMR. This is especially true for highly flexible intrinsically disordered proteins (IDPs) and highly flexible regions (IDRs) of complex multidomain proteins. Indeed, the investigation of these proteins is frequently hampered by the crowding of NMR spectra. The advantages, however, are accompanied by challenges that the user must overcome when conducting experiments at such a high field (e.g., large spectral widths, radio frequency bandwidth, performance of decoupling schemes). This protocol presents strategies and tricks for optimising high-field NMR experiments for IDPs/IDRs based on the analysis of the relaxation properties of the investigated protein. The protocol, tested on three IDPs of different molecular weight and structural complexity, focuses on 13C-detected NMR at 1.2 GHz. A set of experiments, including some multiple receiver experiments, and tips to implement versions tailored for IDPs/IDRs are described. However, the general approach and most considerations can also be applied to experiments that acquire 1H or 15N nuclei and to experiments performed at lower field strengths.

Advances in mass spectrometry to unravel the structure and function of protein condensates.

Membrane-less organelles assemble through liquid-liquid phase separation (LLPS) of partially disordered proteins into highly specialized microenvironments. Currently, it is challenging to obtain a clear understanding of the relationship between the structure and function of phase-separated protein assemblies, owing to their size, dynamics and heterogeneity. In this Perspective, we discuss recent advances in mass spectrometry (MS) that offer several promising approaches for the study of protein LLPS. We survey MS tools that have provided valuable insights into other insoluble protein systems, such as amyloids, and describe how they can also be applied to study proteins that undergo LLPS. On the basis of these recent advances, we propose to integrate MS into the experimental workflow for LLPS studies. We identify specific challenges and future opportunities for the analysis of protein condensate structure and function by MS.

The stability of NPM1 oligomers regulated by acidic disordered regions controls the quality of liquid droplets.

The nucleolus is a membrane-less nuclear body that typically forms through the process of liquid-liquid phase separation (LLPS) involving its components. NPM1 drives LLPS within the nucleolus and its oligomer formation and inter-oligomer interactions play a cooperative role in inducing LLPS. However, the molecular mechanism underlaying the regulation of liquid droplet quality formed by NPM1 remains poorly understood. In this study, we demonstrate that the N-terminal and central acidic residues within the intrinsically disordered regions (IDR) of NPM1 contribute to attenuating oligomer stability, although differences in the oligomer stability were observed only under stringent conditions. Furthermore, the impact of the IDRs is augmented by an increase in net negative charges resulting from phosphorylation within the IDRs. Significantly, we observed an increase in fluidity of liquid droplets formed by NPM1 with decreased oligomer stability. These results indicate that the difference in oligomer stability only observed biochemically under stringent conditions has a significant impact on liquid droplet quality formed by NPM1. Our findings provide new mechanistic insights into the regulation of nucleolar dynamics during the cell cycle.

Nucleic Acids Modulate Liquidity and Dynamics of Artificial Membraneless Organelles.

Liquid-liquid phase separation (LLPS) emerges as a fundamental underlying mechanism for the biological organization, especially the formation of membraneless organelles (MLOs) hosting intrinsically disordered proteins (IDPs) as scaffolds. Nucleic acids are compositional biomacromolecules of MLOs with wide implications in normal cell functions as well as in pathophysiology caused by aberrant phase behavior. Exploiting a minimalist artificial membraneless organelles (AMLO) from LLPS of IDP-mimicking polymer-oligopeptide hybrid (IPH), we investigated the effect of nucleic acids with different lengths and sequence variations on AMLO. The behavior of this AMLO in the presence of DNAs and RNAs resembled natural MLOs in multiple aspects, namely, modulated propensity of formation, morphology, liquidity, and dynamics. Both DNA and RNA could enhance the LLPS of AMLO, while compared with RNA, DNA had a higher tendency to solidify and diminish dynamics thereof. These findings suggest its potential as a concise model system for the understanding of the interaction between nucleic acids and natural MLOs and for studying the molecular mechanism of diseases involving MLOs.

Unambiguous Tracking of Protein Phosphorylation by Fast High-Resolution FOSY NMR*.

Dysregulation of post-translational modifications (PTMs) like phosphorylation is often involved in disease. NMR may elucidate exact loci and time courses of PTMs at atomic resolution and near-physiological conditions but requires signal assignment to individual atoms. Conventional NMR methods for this base on tedious global signal assignment that may often fail, as for large intrinsically disordered proteins (IDPs). We present a sensitive, robust alternative to rapidly obtain only the local assignment near affected signals, based on FOcused SpectroscopY (FOSY) experiments using selective polarisation transfer (SPT). We prove its efficiency by identifying two phosphorylation sites of glycogen synthase kinase 3 beta (GSK3ß) in human Tau40, an IDP of 441 residues, where the extreme spectral dispersion in FOSY revealed unprimed phosphorylation also of Ser409. FOSY may broadly benefit NMR studies of PTMs and other hotspots in IDPs, including sites involved in molecular interactions.

RFPR-IDP: reduce the false positive rates for intrinsically disordered protein and region prediction by incorporating both fully ordered proteins and disordered proteins.

As an important type of proteins, intrinsically disordered proteins/regions (IDPs/IDRs) are related to many crucial biological functions. Accurate prediction of IDPs/IDRs is beneficial to the prediction of protein structures and functions. Most of the existing methods ignore the fully ordered proteins without IDRs during training and test processes. As a result, the corresponding predictors prefer to predict the fully ordered proteins as disordered proteins. Unfortunately, these methods were only evaluated on datasets consisting of disordered proteins without or with only a few fully ordered proteins, and therefore, this problem escapes the attention of the researchers. However, most of the newly sequenced proteins are fully ordered proteins in nature. These predictors fail to accurately predict the ordered and disordered proteins in real-world applications. In this regard, we propose a new method called RFPR-IDP trained with both fully ordered proteins and disordered proteins, which is constructed based on the combination of convolution neural network (CNN) and bidirectional long short-term memory (BiLSTM). The experimental results show that although the existing predictors perform well for predicting the disordered proteins, they tend to predict the fully ordered proteins as disordered proteins. In contrast, the RFPR-IDP predictor can correctly predict the fully ordered proteins and outperform the other 10 state-of-the-art methods when evaluated on a test dataset with both fully ordered proteins and disordered proteins. The web server and datasets of RFPR-IDP are freely available at http://bliulab.net/RFPR-IDP/server.

Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction.

Cryogenic electron microscopy (cryo-EM) is widely used to study biological macromolecules that comprise regions with disorder, flexibility or partial occupancy. For example, membrane proteins are often kept in solution with detergent micelles and lipid nanodiscs that are locally disordered. Such spatial variability negatively impacts computational three-dimensional (3D) reconstruction with existing iterative refinement algorithms that assume rigidity. We introduce non-uniform refinement, an algorithm based on cross-validation optimization, which automatically regularizes 3D density maps during refinement to account for spatial variability. Unlike common shift-invariant regularizers, non-uniform refinement systematically removes noise from disordered regions, while retaining signal useful for aligning particle images, yielding dramatically improved resolution and 3D map quality in many cases. We obtain high-resolution reconstructions for multiple membrane proteins as small as 100 kDa, demonstrating increased effectiveness of cryo-EM for this class of targets critical in structural biology and drug discovery. Non-uniform refinement is implemented in the cryoSPARC software package.

Current Challenges and Limitations in the Studies of Intrinsically Disordered Proteins in Neurodegenerative Diseases by Computer Simulations.

Experiments face challenges in the analysis of intrinsically disordered proteins in solution due to fast conformational changes and enhanced aggregation propensity. Computational studies complement experiments, being widely used in the analyses of intrinsically disordered proteins, especially those positioned at the centers of neurodegenerative diseases. However, recent investigations - including our own - revealed that computer simulations face significant challenges and limitations themselves. In this review, we introduced and discussed some of the scientific challenges and limitations of computational studies conducted on intrinsically disordered proteins. We also outlined the importance of future developments in the areas of computational chemistry and computational physics that would be needed for generating more accurate data for intrinsically disordered proteins from computer simulations. Additional theoretical strategies that can be developed are discussed herein.

Power of Pure Shift HαCα Correlations: A Way to Characterize Biomolecules under Physiological Conditions.

Intrinsically disordered proteins (IDPs) constitute an important class of biomolecules with high flexibility. Atomic-resolution studies for these molecules are essentially limited to NMR spectroscopy, which should be performed under physiological pH and temperature to populate relevant conformational ensembles. In this context, however, fundamental problems arise with established triple resonance NMR experiments: high solvent accessibility of IDPs promotes water exchange, which disfavors classical amide 1H-detection, while 13C-detection suffers from significantly reduced sensitivity. A favorable alternative, the conventional detection of nonexchangeable 1Hα, so far resulted in broad signals with insufficient resolution and sensitivity. To overcome this, we introduce here a selective Hα,Cα-correlating pure shift detection scheme, the selective Hα,Cα-HSQC (SHACA-HSQC), using extensive hetero- and homonuclear decoupling applicable to aqueous samples (≥90% H2O) and tested on small molecules and proteins. SHACA-HSQC spectra acquired on IDPs provide uncompromised resolution and sensitivity (up to fivefold increased S/N compared to the standard 1H,13C-HSQC), as shown for resonance distinction and unambiguous assignment on the disordered transactivation domain of the tumor suppressor p53, α-synuclein, and folded ubiquitin. The detection scheme can be implemented in any 1Hα-detected triple resonance experiment and may also form the basis for the detection of isotope-labeled markers in biological studies or compound libraries.

Detection of Multisite Phosphorylation of Intrinsically Disordered Proteins Using Phos-tag SDS-PAGE.

Phos-tagTM SDS-PAGE is a method that enables electrophoretic separation of proteins based on their phosphorylation status. With Phos-tagTM SDS-PAGE, it is possible to discriminate between different phosphoforms of proteins based on their phosphorylation level and the number of phosphorylated sites, and to determine the stoichiometry of different phosphorylation products. Phos-tagTM SDS-PAGE is useful for analyzing disordered proteins with multiple phosphorylation sites and can be used for any of the downstream applications used in combination with conventional SDS-PAGE, for example, Western blotting and mass-spectrometry. To obtain the best results with Phos-tagTM SDS-PAGE, however, it is often necessary to optimize the gel composition. Depending on the molecular weight and number of phosphoryl groups added to the protein, different gel composition or running conditions should be used. Here, we provide protocols for Mn2+- and Zn2+-Phos-tagTM SDS-PAGE and give examples of how disordered proteins with different characteristics behave in gels with various Phos-tag concentrations.

Detection of Multisite Phosphorylation of Intrinsically Disordered Proteins Using Quantitative Mass-Spectrometry.

Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) within proteins have attracted considerable attention in recent years. Several important biological signaling mechanisms including protein-protein interactions and post-translational modifications can be easily mediated by IDPs and IDRs due to their flexible structure. These regions can encode linear sequences that are indispensable in cell-signaling networks and circuits. For example, the linear multisite phosphorylation networks encoded in disordered protein sequences play a key role in cell-cycle regulation where the phosphorylation of proteins controls the orchestration of all major mechanisms. While elucidating a systems-level understanding of this process and other multisite phosphorylation processes, we extensively used mass-spectrometry and found it to be an ideal tool to identify, characterize, and quantify phosphorylation dynamics within IDPs. Here, we describe a quantitative proteomics method, together with a detailed protocol to analyze dynamic multisite phosphorylation processes within IDPs using an in vitro protein phosphorylation assay with "light" gamma-16O ATP and "heavy" gamma-18O ATP, combined with liquid chromatography mass spectrometry.

An analytical theory to describe sequence-specific inter-residue distance profiles for polyampholytes and intrinsically disordered proteins.

Intrinsically Disordered Proteins (IDPs), unlike folded proteins, lack a unique folded structure and rapidly interconvert among ensembles of disordered states. However, they have specific conformational properties when averaged over their ensembles of disordered states. It is critical to develop a theoretical formalism to predict these ensemble average conformational properties that are encoded in the IDP sequence (the specific order in which amino acids/residues are linked). We present a general heteropolymer theory that analytically computes the ensemble average distance profiles (⟨Rij 2⟩) between any two (i, j) monomers (amino acids for IDPs) as a function of the sequence. Information rich distance profiles provide a detailed description of the IDP in contrast to typical metrics such as scaling exponents, radius of gyration, or end-to-end distance. This generalized formalism supersedes homopolymer-like models or models that are built only on the composition of amino acids but ignore sequence details. The prediction of these distance profiles for highly charged polyampholytes and naturally occurring IDPs unmasks salient features that are hidden in the sequence. Moreover, the model reveals strategies to modulate the entire distance map to achieve local or global swelling/compaction by subtle changes/modifications-such as phosphorylation, a biologically relevant process-in specific hotspots in the sequence. Sequence-specific distance profiles and their modulation have been benchmarked against all-atom simulations. Our new formalism also predicts residue-pair specific coil-globule transitions. The analytical nature of the theory will facilitate design of new sequences to achieve specific target distance profiles with broad applications in synthetic biology and polymer science.

Ratiometric Single-Molecule FRET Measurements to Probe Conformational Subpopulations of Intrinsically Disordered Proteins.

Over the past few decades, numerous examples have demonstrated that intrinsic disorder in proteins lies at the heart of many vital processes, including transcriptional regulation, stress response, cellular signaling, and most recently protein liquid-liquid phase separation. The so-called intrinsically disordered proteins (IDPs) involved in these processes have presented a challenge to the classic protein "structure-function paradigm," as their functions do not necessarily involve well-defined structures. Understanding the mechanisms of IDP function is likewise challenging because traditional structure determination methods often fail with such proteins or provide little information about the diverse array of structures that can be related to different functions of a single IDP. Single-molecule fluorescence methods can overcome this ensemble-average masking, allowing the resolution of subpopulations and dynamics and thus providing invaluable insights into IDPs and their function. In this protocol, we describe a ratiometric single-molecule Förster resonance energy transfer (smFRET) routine that permits the investigation of IDP conformational subpopulations and dynamics. We note that this is a basic protocol, and we provide brief information and references for more complex analysis schemes available for in-depth characterization. This protocol covers optical setup preparation and protein handling and provides insights into experimental design and outcomes, together with background information about theory and a brief discussion of troubleshooting. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Ratiometric smFRET detection and analysis of IDPs Support Protocol 1: Fluorophore labeling of a protein through maleimide chemistry Support Protocol 2: Sample chamber preparation Support Protocol 3: Determination of direct excitation of acceptor by donor excitation and leakage of donor emission to acceptor emission channel.

Proteome-wide analysis of protein disorder in Triticum aestivum and Hordeum vulgare.

There has been an increasing interest in Intrinsically Disordered Proteins (IDPs) ever since it was proven that they are ubiquitous and involved in key cellular functions. Interestingly, they have shown a large abundance in complete proteomes. In the current study, we have investigated the first large-scale study of the repertoire of IDPs in Triticum aestivum and Hordeum vulgare proteomes, in order to get insight into the biological roles of IDPs in both species. Results show that proteins in T. aestivum are significantly more disordered than those of H. vulgare. Moreover, the data revealed that DNA/RNA binding domains, co-factors, heme, metal ions binding domains, ATP/GTP binding proteins, ligands, linker domains and repeats, other domains typical to transcription factors such as zinc finger, F-box domain, homeodomain-like, l-domain like and chaperones, are predominantly present and co-occur in disordered proteins in T.aestivum and H.vulgare. The Gene Ontology analysis revealed that IDPs in T. aestivum and H. vulgare are mainly involved in regulation of cellular and biological processes up on response to stress. In future, this study may provide valuable information while considering IDPs in understanding the organism complexity and environmental adaptation.

Functional Segments on Intrinsically Disordered Regions in Disease-Related Proteins.

One of the unique characteristics of intrinsically disordered proteins (IPDs) is the existence of functional segments in intrinsically disordered regions (IDRs). A typical function of these segments is binding to partner molecules, such as proteins and DNAs. These segments play important roles in signaling pathways and transcriptional regulation. We conducted bioinformatics analysis to search these functional segments based on IDR predictions and database annotations. We found more than a thousand potential functional IDR segments in disease-related proteins. Large fractions of proteins related to cancers, congenital disorders, digestive system diseases, and reproductive system diseases have these functional IDRs. Some proteins in nervous system diseases have long functional segments in IDRs. The detailed analysis of some of these regions showed that the functional segments are located on experimentally verified IDRs. The proteins with functional IDR segments generally tend to come and go between the cytoplasm and the nucleus. Proteins involved in multiple diseases tend to have more protein-protein interactors, suggesting that hub proteins in the protein-protein interaction networks can have multiple impacts on human diseases.

NMR Characterization of Long-Range Contacts in Intrinsically Disordered Proteins from Paramagnetic Relaxation Enhancement in 13 C Direct-Detection Experiments.

Intrinsically disordered proteins (IDPs) carry out many biological functions. They lack a stable 3D structure and are able to adopt many different conformations in dynamic equilibrium. The interplay between local dynamics and global rearrangements is key for their function. A widely used experimental NMR spectroscopy approach to study long-range contacts in IDPs exploits paramagnetic effects, and 1 H detection experiments are generally used to determine paramagnetic relaxation enhancement (PRE) for amide protons. However, under physiological conditions, exchange broadening hampers the detection of solvent-exposed amide protons, which reduces the content of information available. Herein, we present an experimental approach based on direct carbon detection of PRE that provides improved resolution, reduced sensitivity to exchange broadening, and complementary information derived from the use of different starting polarization sources.

Challenges in the Structural-Functional Characterization of Multidomain, Partially Disordered Proteins CBP and p300: Preparing Native Proteins and Developing Nanobody Tools.

The structural and functional characterization of large multidomain signaling proteins containing long disordered linker regions represents special methodological and conceptual challenges. These proteins show extreme structural heterogeneity and have complex posttranslational modification patterns, due to which traditional structural biology techniques provide results that are often difficult to interpret. As demonstrated through the example of two such multidomain proteins, CREB-binding protein (CBP) and its paralogue, p300, even the expression and purification of such proteins are compromised by their extreme proteolytic sensitivity and structural heterogeneity. In this chapter, we describe the effective expression of CBP and p300 in a eukaryotic host, Sf9 insect cells, followed by their tandem affinity purification based on two terminal tags to ensure their structural integrity. The major focus of this chapter is on the development of novel accessory tools, single-domain camelid antibodies (nanobodies), for structural-functional characterization. Specific nanobodies against full-length CBP and p300 can specifically target their different regions and can be used for their marking, labeling, and structural stabilization in a broad range of in vitro and in vivo studies. Here, we describe four high-affinity nanobodies binding to the KIX and the HAT domains, either mimicking known interacting partners or revealing new functionally relevant conformations. As immunization of llamas results in nanobody libraries with a great sequence variation, deep sequencing and interaction analysis with different regions of the proteins provide a novel approach toward developing a panel of specific nanobodies.

High-dimensional NMR methods for intrinsically disordered proteins studies.

Intrinsically disordered proteins (IDPs) are getting more and more interest of the scientific community. Nuclear magnetic resonance (NMR) is often a technique of choice for these studies, as it provides atomic-resolution information on structure, dynamics and interactions of IDPs. Nonetheless, NMR spectra of IDPs are typically extraordinary crowded, comparing to those of structured proteins. To overcome this problem, high-dimensional NMR experiments can be used, which allow for a better peak separation. In the present review different aspects of such experiments are discussed, from data acquisition and processing to analysis, focusing on experiments for resonance assignment.

Deciphering the dark proteome of Chikungunya virus.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus. The outbreak of CHIKV infection has been seen in many tropical and subtropical regions of the biosphere. Current reports evidenced that after outbreaks in 2005-06, the fitness of this virus propagating in Aedes albopictus enhanced due to the epistatic mutational changes in its envelope protein. In our study, we evaluated the prevalence of intrinsically disordered proteins (IDPs) and IDP regions (IDPRs) in CHIKV proteome. IDPs/IDPRs are known as members of a 'Dark Proteome' that defined as a set of polypeptide segments or whole protein without unique three-dimensional structure within the cellular milieu but with significant biological functions, such as cell cycle regulation, control of signaling pathways, and maintenance of viral proteomes. However, the intrinsically disordered aspects of CHIKV proteome and roles of IDPs/IDPRs in the pathogenic mechanism of this important virus have not been evaluated as of yet. There are no existing reports on the analysis of intrinsic disorder status of CHIKV. To fulfil this goal, we have analyzed the abundance and functionality of IDPs/IDPRs in CHIKV proteins, involved in the replication and maturation. It is likely that these IDPs/IDPRs can serve as novel targets for disorder based drug design.

MINAR1 is a Notch2-binding protein that inhibits angiogenesis and breast cancer growth.

Intrinsically disordered proteins (IDPs)/intrinsically unstructured proteins are characterized by the lack of fixed or stable tertiary structure, and are increasingly recognized as an important class of proteins with major roles in signal transduction and transcriptional regulation. In this study, we report the identification and functional characterization of a previously uncharacterized protein (UPF0258/KIAA1024), major intrinsically disordered Notch2-associated receptor 1 (MINAR1). While MINAR1 carries a single transmembrane domain and a short cytoplasmic domain, it has a large extracellular domain that shares no similarity with known protein sequences. Uncharacteristically, MINAR1 is a highly IDP with nearly 70% of its amino acids sequences unstructured. We demonstrate that MINAR1 physically interacts with Notch2 and its binding to Notch2 increases its stability and function. MINAR1 is widely expressed in various tissues including the epithelial cells of the breast and endothelial cells of blood vessels. MINAR1 plays a negative role in angiogenesis as it inhibits angiogenesis in cell culture and in mouse matrigel plug and zebrafish angiogenesis models. Furthermore, while MINAR1 is highly expressed in the normal human breast, its expression is significantly downregulated in advanced human breast cancer and its re-expression in breast cancer cells inhibited tumor growth. Our study demonstrates that MINAR1 is an IDP that negatively regulates angiogenesis and growth of breast cancer cells.