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.

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.

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.

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.

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.

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.

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.

Direct detection of carbon and nitrogen nuclei for high-resolution analysis of intrinsically disordered proteins using NMR spectroscopy.

Nuclear magnetic resonance spectroscopy (NMR) is a powerful technique for characterizing the structural and dynamic properties of intrinsically disordered proteins and protein regions (IDPs & IDRs). However, the application of NMR to IDPs has been limited by poor chemical shift dispersion in two-dimensional (2D) 1H-15N heteronuclear correlation spectra. Among the various detection schemes available for heteronuclear correlation spectroscopy, 13C direct-detection has become a mainstay for investigations of IDPs owing to the favorable chemical shift dispersion in 2D 13C'-15N correlation spectra. Recent advances in cryoprobe technology have enhanced the sensitivity for direct detection of both 13C and 15N resonances at high magnetic field strengths, thus prompting the development of 15N direct-detect experiments to complement established 13C-detection experiments. However, the application of 15N-detection has not been widely explored for IDPs. Here we compare 1H, 13C, and 15N detection schemes for a variety of 2D heteronuclear correlation spectra and evaluate their performance on the basis of resolution, chemical shift dispersion, and sensitivity. We performed experiments with a variety of disordered systems ranging in size and complexity; from a small IDR (99 amino acids), to a large low complexity IDR (185 amino acids), and finally a ∼73 kDa folded homopentameric protein that also contains disordered regions (133 amino acids/monomer). We conclude that, while requiring high sample concentration and long acquisition times, 15N-detection often offers enhanced resolution over other detection schemes in studies of disordered protein regions with low complexity sequences.

Relating gas phase to solution conformations: Lessons from disordered proteins.

In recent years both mass spectrometry (MS) and ion mobility mass spectrometry (IM-MS) have been developed as techniques with which to study proteins that lack a fixed tertiary structure but may contain regions that form secondary structure elements transiently, namely intrinsically disordered proteins (IDPs). IM-MS is a suitable method for the study of IDPs which provides an insight to conformations that are present in solution, potentially enabling the analysis of lowly populated structural forms. Here, we describe the IM-MS data of two IDPs; α-Synuclein (α-Syn) which is implicated in Parkinson's disease, and Apolipoprotein C-II (ApoC-II) which is involved in cardiovascular diseases. We report an apparent discrepancy in the way that ApoC-II behaves in the gas phase. While most IDPs, including α-Syn, present in many charge states and a wide range of rotationally averaged collision cross sections (CCSs), ApoC-II presents in just four charge states and a very narrow range of CCSs, independent of solution conditions. Here, we compare MS and IM-MS data of both proteins, and rationalise the differences between the proteins in terms of different ionisation processes which they may adhere to.

Easy and unambiguous sequential assignments of intrinsically disordered proteins by correlating the backbone 15N or 13C' chemical shifts of multiple contiguous residues in highly resolved 3D spectra.

Sequential resonance assignment strategies are typically based on matching one or two chemical shifts of adjacent residues. However, resonance overlap often leads to ambiguity in resonance assignments in particular for intrinsically disordered proteins. We investigated the potential of establishing connectivity through the three-bond couplings between sequentially adjoining backbone carbonyl carbon nuclei, combined with semi-constant time chemical shift evolution, for resonance assignments of small folded and larger unfolded proteins. Extended sequential connectivity strongly lifts chemical shift degeneracy of the backbone nuclei in disordered proteins. We show here that 3D (H)N(COCO)NH and (HN)CO(CO)NH experiments with relaxation-optimized multiple pulse mixing correlate up to seven adjacent backbone amide nitrogen or carbonyl carbon nuclei, respectively, and connections across proline residues are also obtained straightforwardly. Multiple, recurrent long-range correlations with ultra-high resolution allow backbone (1)H(N), (15)N(H), and (13)C' resonance assignments to be completed from a single pair of 3D experiments.

Spectral density mapping protocols for analysis of molecular motions in disordered proteins.

Spectral density mapping represents the method of choice for investigations of molecular motions of intrinsically disordered proteins (IDPs). However, the current methodology has been developed for well-folded proteins. In order to find conditions for a reliable analysis of relaxation of IDPs, accuracy of the current reduced spectral density mapping protocols applied to IDPs was examined and new spectral density mapping methods employing cross-correlated relaxation rates have been designed. Various sources of possible systematic errors were analyzed theoretically and the presented approaches were tested on a partially disordered protein, delta subunit of bacterial RNA polymerase. Results showed that the proposed protocols provide unbiased description of molecular motions of IDPs and allow to separate slow exchange from fast dynamics.

CP-HISQC: a better version of HSQC experiment for intrinsically disordered proteins under physiological conditions.

(1)H-(15)N HSQC spectroscopy is a workhorse of protein NMR. However, under physiological conditions the quality of HSQC spectra tends to deteriorate due to fast solvent exchange. For globular proteins only a limited number of surface residues are affected, but in the case of intrinsically disordered proteins (IDPs) HSQC spectra are thoroughly degraded, suffering from both peak broadening and loss of intensity. To alleviate this problem, we make use of the following two concepts. (1) Proton-decoupled HSQC. Regular HSQC and its many variants record the evolution of multi-spin modes, 2NxHz or 2NxHx, in indirect dimension. Under the effect of fast solvent exchange these modes undergo rapid decay, which results in severe line-broadening. In contrast, proton-decoupled HSQC relies on Nx coherence which is essentially insensitive to the effects of solvent exchange. Moreover, for measurements involving IDPs at or near physiological temperature, Nx mode offers excellent relaxation properties, leading to very sharp resonances. (2) Cross-polarization (1)H-to-(15)N transfer. If CP element is designed such as to lock both (1)H(N) and water magnetization, the following transfer is effected: [Formula: see text] Thus water magnetization is successfully exploited to boost the amount of signal. In addition, CP element suffers less loss from solvent exchange, conformational exchange, and dipolar relaxation compared to the more popular INEPT element. Combining these two concepts, we have implemented the experiment termed CP-HISQC (cross-polarization assisted heteronuclear in-phase single-quantum correlation). The pulse sequence has been designed such as to preserve water magnetization and therefore can be executed with reasonably short recycling delays. In the presence of fast solvent exchange, kex ~ 100 s(-1), CP-HISQC offers much better spectral resolution than conventional HSQC-type experiments. At the same time it offers up to twofold gain in sensitivity compared to plain proton-decoupled HSQC. The new sequence has been tested on the sample of drkN SH3 domain at pH 7.5, 30 °C. High-quality spectrum has been recorded in less than 1 h, containing resonances from both folded and unfolded species. High-quality spectra have also been obtained for arginine side-chain H(ε)N(ε) groups in the sample of short peptide Sos. For Arg side chains, we have additionally implemented (HE)NE(CD)HD experiment. Using (13)C-labeled sample of Sos, we have demonstrated that proton-to-nitrogen CP transfer remains highly efficient in the presence of solvent exchange as fast as kex = 620 s(-1). In contrast, INEPT transfer completely fails in this regime.

Generating NMR chemical shift assignments of intrinsically disordered proteins using carbon-detected NMR methods.

There is an extraordinary need to describe the structures of intrinsically disordered proteins (IDPs) due to their role in various biological processes involved in signaling and transcription. However, general study of IDPs by NMR spectroscopy is limited by the poor (1)H amide chemical shift dispersion typically observed in their spectra. Recently, (13)C direct-detected NMR spectroscopy has been recognized as enabling broad structural study of IDPs. Most notably, multidimensional experiments based on the (15)N,(13)C CON spectrum make complete chemical shift assignment feasible. Here we document a collection of NMR-based tools that efficiently lead to chemical shift assignment of IDPs, motivated by a case study of the C-terminal disordered region from the human pancreatic transcription factor Pdx1. Our strategy builds on the combination of two three-dimensional (3D) experiments, (HN-flip)N(CA)CON and 3D (HN-flip)N(CA)NCO, that enable daisy chain connections to be built along the IDP backbone, facilitated by acquisition of amino acid-specific (15)N,(13)C CON-detected experiments. Assignments are completed through carbon-detected, total correlation spectroscopy (TOCSY)-based side chain chemical shift measurement. Conducting our study required producing valuable modifications to many previously published pulse sequences, motivating us to announce the creation of a database of our pulse programs, which we make freely available through our website.

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.

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.

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.

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.

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.

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.