Minimum information guidelines for experiments structurally characterizing intrinsically disordered protein regions.

An unambiguous description of an experiment, and the subsequent biological observation, is vital for accurate data interpretation. Minimum information guidelines define the fundamental complement of data that can support an unambiguous conclusion based on experimental observations. We present the Minimum Information About Disorder Experiments (MIADE) guidelines to define the parameters required for the wider scientific community to understand the findings of an experiment studying the structural properties of intrinsically disordered regions (IDRs). MIADE guidelines provide recommendations for data producers to describe the results of their experiments at source, for curators to annotate experimental data to community resources and for database developers maintaining community resources to disseminate the data. The MIADE guidelines will improve the interpretability of experimental results for data consumers, facilitate direct data submission, simplify data curation, improve data exchange among repositories and standardize the dissemination of the key metadata on an IDR experiment by IDR data sources.

Are Protein Conformational Ensembles in Agreement with Experimental Data? A Geometrical Interpretation of the Problem.

The conformational variability of biological macromolecules can play an important role in their biological function. Therefore, understanding conformational variability is expected to be key for predicting the behavior of a particular molecule in the context of organism-wide studies. Several experimental methods have been developed and deployed for accessing this information, and computational methods are continuously updated for the profitable integration of different experimental sources. The outcome of this endeavor is conformational ensembles, which may vary significantly in properties and composition when different ensemble reconstruction methods are used, and this raises the issue of comparing the predicted ensembles against experimental data. In this article, we discuss a geometrical formulation to provide a framework for understanding the agreement of an ensemble prediction to the experimental observations.

13C Direct Detected NMR for Challenging Systems.

Thanks to recent improvements in NMR spectrometer hardware and pulse sequence design, modern 13C NMR has become a useful tool for biomolecular applications. The complete assignment of a protein can be accomplished by using 13C detected multinuclear experiments and it can provide unique information relevant for the study of a variety of different biomolecules including paramagnetic proteins and intrinsically disordered proteins. A wide range of NMR observables can be measured, concurring to the structural and dynamic characterization of a protein in isolation, as part of a larger complex, or even inside a living cell. We present the different properties of 13C with respect to 1H, which provide the rationale for the experiments developed and their application, the technical aspects that need to be faced, and the many experimental variants designed to address different cases. Application areas where these experiments successfully complement proton NMR are also described.

Picometer Resolution Structure of the Coordination Sphere in the Metal-Binding Site in a Metalloprotein by NMR.

Most of our understanding of chemistry derives from atomic-level structures obtained with single-crystal X-ray diffraction. Metal centers in X-ray structures of small organometallic or coordination complexes are often extremely well-defined, with errors in the positions on the order of 10-4-10-5 Å. Determining the metal coordination geometry to high accuracy is essential for understanding metal center reactivity, as even small structural changes can dramatically alter the metal activity. In contrast, the resolution of X-ray structures in proteins is limited typically to the order of 10-1 Å. This resolution is often not sufficient to develop precise structure-activity relations for the metal sites in proteins, because the uncertainty in positions can cover all of the known ranges of bond lengths and bond angles for a given type of metal complex. Here we introduce a new approach that enables the determination of a high-definition structure of the active site of a metalloprotein from a powder sample, by combining magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, tailored radio frequency (RF) irradiation schemes, and computational approaches. This allows us to overcome the "blind sphere" in paramagnetic proteins, and to observe and assign 1H, 13C, and 15N resonances for the ligands directly coordinating the metal center. We illustrate the method by determining the bond lengths in the structure of the CoII coordination sphere at the core of human superoxide dismutase 1 (SOD) with 0.7 pm precision. The coordination geometry of the resulting structure explains the nonreactive nature of the CoII/ZnII centers in these proteins, which allows them to play a purely structural role.

Multimodal Response to Copper Binding in Superoxide Dismutase Dynamics.

Copper/zinc superoxide dismutase (SOD) is a homodimeric metalloenzyme that has been extensively studied as a benchmark for structure-function relationships in proteins, in particular because of its implication in the familial form of the neurodegenerative disease amyotrophic lateral sclerosis. Here, we investigate microcrystalline preparations of two differently metalated forms of SOD, namely, the fully mature functional Cu,Zn state and the E,Zn-SOD state in which the Cu site is empty. By using solid-state NMR with fast magic-angle spinning (MAS) at high magnetic fields (1H Larmor frequency of 800-1000 MHz), we quantify motions spanning a dynamic range from ns to ms. We determine that metal ion uptake does not act as a rigidification element but as a switch redistributing motional processes on different time scales, with coupling of the dynamics of histidine side chains and those of remote key backbone elements of the protein.

Sensitivity-enhanced three-dimensional and carbon-detected two-dimensional NMR of proteins using hyperpolarized water.

Signal enhancements of up to two orders of magnitude in protein NMR can be achieved by employing HDO as a vector to introduce hyperpolarization into folded or intrinsically disordered proteins. In this approach, hyperpolarized HDO produced by dissolution-dynamic nuclear polarization (D-DNP) is mixed with a protein solution waiting in a high-field NMR spectrometer, whereupon amide proton exchange and nuclear Overhauser effects (NOE) transfer hyperpolarization to the protein and enable acquisition of a signal-enhanced high-resolution spectrum. To date, the use of this strategy has been limited to 1D and 1H-15N 2D correlation experiments. Here we introduce 2D 13C-detected D-DNP, to reduce exchange-induced broadening and other relaxation penalties that can adversely affect proton-detected D-DNP experiments. We also introduce hyperpolarized 3D spectroscopy, opening the possibility of D-DNP studies of larger proteins and IDPs, where assignment and residue-specific investigation may be impeded by spectral crowding. The signal enhancements obtained depend in particular on the rates of chemical and magnetic exchange of the observed residues, thus resulting in non-uniform 'hyperpolarization-selective' signal enhancements. The resulting spectral sparsity, however, makes it possible to resolve and monitor individual amino acids in IDPs of over 200 residues at acquisition times of just over a minute. We apply the proposed experiments to two model systems: the compactly folded protein ubiquitin, and the intrinsically disordered protein (IDP) osteopontin (OPN).

Monitoring the Interaction of α-Synuclein with Calcium Ions through Exclusively Heteronuclear Nuclear Magnetic Resonance Experiments.

Many properties of intrinsically disordered proteins (IDPs), or protein regions (IDRs), are modulated by the nature of amino acid side chains as well as by local solvent exposure. We propose a set of exclusively heteronuclear NMR experiments to investigate these features in different experimental conditions that are relevant for physiological function. The proposed approach is generally applicable to many IDPs/IDRs whose assignment is available in the Biological Magnetic Resonance Bank (BMRB) to investigate how their properties are modulated by different, physiologically relevant conditions. The experiments, tested on α-synuclein, are then used to investigate how α-synuclein senses Ca2+ concentration jumps associated with the transmission of nerve signals. Novel modules in the primary sequence of α-synuclein optimized for calcium sensing in highly flexible, disordered protein segments are identified.

Protein NMR Resonance Assignment without Spectral Analysis: 5D SOlid-State Automated Projection SpectroscopY (SO-APSY).

Narrow proton signals, high sensitivity, and efficient coherence transfers provided by fast magic-angle spinning at high magnetic fields make automated projection spectroscopy feasible for the solid-state NMR analysis of proteins. We present the first ultrahigh dimensional implementation of this approach, where 5D peak lists are reconstructed from a number of 2D projections for protein samples of different molecular sizes and aggregation states, which show limited dispersion of chemical shifts or inhomogeneous broadenings. The resulting datasets are particularly suitable to automated analysis and yield rapid and unbiased assignments of backbone resonances.

Taking Simultaneous Snapshots of Intrinsically Disordered Proteins in Action.

Intrinsically disordered proteins (IDPs) as well as intrinsically disordered regions (IDRs) of complex protein machineries have recently been recognized as key players in many cellular functions. NMR represents a unique tool to access atomic resolution structural and dynamic information on highly flexible IDPs/IDRs. Improvements in instrumental sensitivity made heteronuclear direct detection possible for biomolecular NMR applications. The CON experiment has become one of the most useful NMR experiments to get a snapshot of an IDP/IDR in conditions approaching physiological ones. The availability of NMR spectrometers equipped with multiple receivers now enables the acquisition of several experiments simultaneously instead of one after the other. Here, we propose several variants of the CON experiment in which, during the recovery delay, a second two-dimensional experiment is acquired, either based on 1H detection (CON//HN) or on 15N detection (CON//btNH, CON//(H)CAN). The possibility to collect simultaneous snapshots of an IDP/IDR through different two-dimensional spectra provides a novel tool to follow chemical reactions, such as the occurrence of posttranslational modifications, as well as to study samples of limited lifetime such as cell lysates or whole cells.

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.

13C APSY-NMR for sequential assignment of intrinsically disordered proteins.

The increasingly recognized biological relevance of intrinsically disordered proteins requires a continuous expansion of the tools for their characterization via NMR spectroscopy, the only technique so far able to provide atomic-resolution information on these highly mobile macromolecules. Here we present the implementation of projection spectroscopy in 13C-direct detected NMR experiments to achieve the sequence specific assignment of IDPs. The approach was used to obtain the complete backbone assignment at high temperature of α-synuclein, a paradigmatic intrinsically disordered protein.

High-Resolution 2D NMR of Disordered Proteins Enhanced by Hyperpolarized Water.

This study demonstrates the usefulness derived from relying on hyperpolarized water obtained by dissolution DNP, for site-resolved biophysical NMR studies of intrinsically disordered proteins. Thanks to the facile amide-solvent exchange experienced by protons in these proteins, 2D NMR experiments that like HMQC rely on the polarization of the amide protons, can be enhanced using hyperpolarized water by several orders of magnitude over their conventional counterparts. Optimizations of the DNP procedure and of the subsequent injection into the protein sample are necessary to achieve these gains while preserving state-of-the-art resolution; procedures enabling this transfer of the hyperpolarized water and the achievement of foamless hyperpolarized protein solutions are demonstrated. These protocols are employed to collect 2D 15N-1H HMQC NMR spectra of α-synuclein, showing residue-specific enhancements ≥100× over their thermal counterparts. These enhancements, however, vary considerably throughout the residues. The biophysics underlying this residue-specific behavior upon injection of hyperpolarized water is theoretically examined, the information that it carries is compared with results arising from alternative methods, and its overall potential is discussed.

Proline Fingerprint in Intrinsically Disordered Proteins.

NMR spectroscopy is one of the main techniques used for high-resolution studies of intrinsically disordered proteins (IDPs), permitting mapping of the structural and dynamic features of all the amino acids constituting the polypeptide at atomic resolution. Only proline residues are less straightforward to characterize because they lack any amide proton, thus rendering them not directly visible in the commonly used 2D 1 H,15 N correlation experiments. However, proline residues are highly abundant in IDPs and can mediate important functions. In this work we present an easy and effective way to obtain fingerprints of proline residues in IDPs at high resolution.

Fragment-Based NMR Study of the Conformational Dynamics in the bHLH Transcription Factor Ascl1.

The Achaete-scute homolog 1 (Ascl1) protein regulates a large subset of genes that leads neuronal progenitor cells to distinctive differentiation pathways during human brain development. Although it is well known that Ascl1 binds DNA as a homo- or heterodimer via its basic helix-loop-helix (bHLH) motif, little is known about the conformational sampling properties of the DNA-free full-length protein, and in particular about the bHLH domain-flanking N- and C-terminal segments, which are predicted to be highly disordered in solution. The structural heterogeneity, low solubility, and high aggregation propensity of Ascl1 in aqueous buffer solutions make high-resolution studies of this protein a challenging task. Here, we have adopted a fragment-based strategy that allowed us to obtain high-quality NMR data providing, to our knowledge, the first comprehensive high-resolution information on the structural propensities and conformational dynamics of Ascl1. The emerging picture is that of an overall extended and highly dynamic polypeptide chain comprising three helical segments and lacking persistent long-range interactions. We also show that the C-terminal helix of the bHLH domain is involved in intermolecular interactions, even in the absence of DNA. Our results contribute to a better understanding of the mechanisms of action that govern the regulation of proneural transcription factors.

Sequence Context Influences the Structure and Aggregation Behavior of a PolyQ Tract.

Expansions of polyglutamine (polyQ) tracts in nine different proteins cause a family of neurodegenerative disorders called polyQ diseases. Because polyQ tracts are potential therapeutic targets for these pathologies there is great interest in characterizing the conformations that they adopt and in understanding how their aggregation behavior is influenced by the sequences flanking them. We used solution NMR to study at single-residue resolution a 156-residue proteolytic fragment of the androgen receptor that contains a polyQ tract associated with the disease spinobulbar muscular atrophy, also known as Kennedy disease. Our findings indicate that a Leu-rich region preceding the polyQ tract causes it to become α-helical and appears to protect the protein against aggregation, which represents a new, to our knowledge, mechanism by which sequence context can minimize the deleterious properties of these repetitive regions. Our results have implications for drug discovery for polyQ diseases because they suggest that the residues flanking these repetitive sequences may represent viable therapeutic targets.

Just a Flexible Linker? The Structural and Dynamic Properties of CBP-ID4 Revealed by NMR Spectroscopy.

Here, we present a structural and dynamic description of CBP-ID4 at atomic resolution. ID4 is the fourth intrinsically disordered linker of CREB-binding protein (CBP). In spite of the largely disordered nature of CBP-ID4, NMR chemical shifts and relaxation measurements show a significant degree of α-helix sampling in the protein regions encompassing residues 2-25 and 101-128 (1852-1875 and 1951-1978 in full-length CBP). Proline residues are uniformly distributed along the polypeptide, except for the two α-helical regions, indicating that they play an active role in modulating the structural features of this CBP fragment. The two helical regions are lacking known functional motifs, suggesting that they represent thus-far uncharacterized functional modules of CBP. This work provides insights into the functions of this protein linker that may exploit its plasticity to modulate the relative orientations of neighboring folded domains of CBP and fine-tune its interactions with a multitude of partners.

Amino acid recognition for automatic resonance assignment of intrinsically disordered proteins.

Resonance assignment is a prerequisite for almost any NMR-based study of proteins. It can be very challenging in some cases, however, due to the nature of the protein under investigation. This is the case with intrinsically disordered proteins, for example, whose NMR spectra suffer from low chemical shifts dispersion and generally low resolution. For these systems, sequence specific assignment is highly time-consuming, so the prospect of using automatic strategies for their assignment is very attractive. In this article we present a new version of the automatic assignment program TSAR dedicated to intrinsically disordered proteins. In particular, we demonstrate how the automatic procedure can be improved by incorporating methods for amino acid recognition and information on chemical shifts in selected amino acids. The approach was tested in silico on 16 disordered proteins and experimentally on α-synuclein, with remarkably good results.

Structural and Dynamic Characterization of the Molecular Hub Early Region 1A (E1A) from Human Adenovirus.

The small-DNA human adenovirus encodes one of the most versatile molecular hubs, the E1A protein. This protein is essential for productive viral infection in human cells and a vast amount of biologically relevant data are available on its interactions with host proteins. Up to now, however, no high-resolution structural and dynamic information on E1A is available despite its important biological role. Among the different spliced variants of E1A, two are expressed at high level in the early stage of infection. These are 243 and 289 residues isoforms. Herein, we present their NMR characterization, showing that they are both highly disordered, but also demonstrate a certain heterogeneous behavior in terms of structural and dynamic properties. Furthermore, we present the characterization of the isolated domain of the longer variant, known as CR3. This study opens the way to understanding at the molecular level how E1A functions.

pE-DB: a database of structural ensembles of intrinsically disordered and of unfolded proteins.

The goal of pE-DB (http://pedb.vib.be) is to serve as an openly accessible database for the deposition of structural ensembles of intrinsically disordered proteins (IDPs) and of denatured proteins based on nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and other data measured in solution. Owing to the inherent flexibility of IDPs, solution techniques are particularly appropriate for characterizing their biophysical properties, and structural ensembles in agreement with these data provide a convenient tool for describing the underlying conformational sampling. Database entries consist of (i) primary experimental data with descriptions of the acquisition methods and algorithms used for the ensemble calculations, and (ii) the structural ensembles consistent with these data, provided as a set of models in a Protein Data Bank format. PE-DB is open for submissions from the community, and is intended as a forum for disseminating the structural ensembles and the methodologies used to generate them. While the need to represent the IDP structures is clear, methods for determining and evaluating the structural ensembles are still evolving. The availability of the pE-DB database is expected to promote the development of new modeling methods and leads to a better understanding of how function arises from disordered states.

The crowd you're in with: effects of different types of crowding agents on protein aggregation.

The intracellular environment contains high concentrations of macromolecules occupying up to 30% of the total cellular volume. Presence of these macromolecules decreases the effective volume available for the proteins in the cell and thus increases the effective protein concentrations and stabilizes the compact protein conformations. Macromolecular crowding created by various macromolecules such as proteins, nucleic acids, and carbohydrates has been shown to have a significant effect on a variety of cellular processes including protein aggregation. Most studies of macromolecular crowding have used neutral, flexible polysaccharides that function primarily via excluded volume effect as model crowding agents. Here we have examined the effects of more rigid polysaccharides on protein structure and aggregation. Our results indicate that rigid and flexible polysaccharides influence protein aggregation via different mechanisms and suggest that, in addition to excluded volume effect, changes in solution viscosity and non-specific protein-polymer interactions influence the structure and dynamics of proteins in crowded environments.