Intrinsically disordered proteins (IDPs) studied by EPR and in-cell EPR

Sabrina Weickert, Julia Cattani and Malte Drescher

DOI: 10.1039/9781788013888-00001

Intrinsically disordered proteins (IDPs) play important physiological, but also disease-related roles. In order to understand the function and malfunction of proteins of this class, electron paramagnetic resonance (EPR) spectroscopy has proven to be a valuable tool, allowing investigation of the protein structural ensembles upon interaction with the environment. This review focuses on the IDPs tau and a-synuclein and gives an overview over recent EPR studies performed with these proteins.

1 Introduction

1.1 IDPs

Intrinsically disordered proteins (IDPs) are a class of proteins lacking a stable three-dimensional structure in solution. Nonetheless, these proteins are native and fulfill many important biological functions, among them cell signaling, recognition and regulation. IDPs are highly prevalent in humans: Genome analyses predict that around 25% of all human proteins are disordered from end to end, while up to 40% contain unstructured regions. Due to their abundance as well as their unique structural and dynamical flexibility, IDPs are key players in many biological pathways, capable of specific interactions with a multitude of binding partners and therefore often binding to or serving as hubs in protein interaction networks. Due to the same reasons, many members of the IDP family are known to be associated with a variety of human diseases, among them prominently cancers, cardiovascular diseases, diabetes and neurodegenerative diseases. All of this has made IDPs a research field of tremendous importance and interest since the turn of the century.

1.1.1 The peculiar free energy landscape of IDPs. In the free energy landscape of proteins with a native globular fold there is a pronounced free energy minimum that stabilizes a distinct 3D fold, which often represents the unique functional form of a globular protein (Fig. 1A). The decrease in entropy associated with restrictions of the conformational freedom during folding is compensated by the formation of many intramolecular contacts. However, the free energy landscape of an IDP looks distinctly different (Fig. 1B): It is characterized by the lack of a global free energy minimum, but shows many local minima instead, which are separated by small energy barriers that allow quick and frequent interconversion between the accessible states. Consequently, the conformational ensemble of an IDP in solution is heterogeneous and characterized by a dynamic exchange between many accessible structures. This non-folding behavior is encoded in the amino acid composition of IDPs: These proteins contain less hydrophobic (Ile, Leu, Val) and aromatic (Trp, Tyr, Phe) amino acid residues, but a significantly larger proportion of small and hydrophilic amino acid residues (Arg, Gly, Gln, Ser, Pro, Glu, Lys) and are richer in structure-breaking amino acid residues (Pro, Gly) than typical globular proteins. Although IDPs cannot spontaneously fold into a compact globular structure, the presence of interaction partners can alter the IDP free energy landscape in a way that more pronounced energy minima appear: Upon interaction with a partner, IDPs often undergo a structural reorganization that defines a state of clearly reduced free energy, which is thermodynamically stabilized (Fig. 1B).

1.1.2 Linking folding and binding. While some IDPs undergo a folding process as a whole upon interaction with a binding partner, more commonly specific recognition motifs in the disordered protein adopt secondary or tertiary structural elements upon interaction with a binding partner. As a mechanism of these disorder-to-order transitions, two major models are discussed in the literature – the 'conformational selection' model and the 'induced folding' model. The first model is based on the assumption that the binding partner selects a specific conformation resembling the bound conformation from the wide ensemble of coexisting conformations the IDP adopts when free in solution (Fig. 1B). The latter model is based on the assumption that the IDP binds to its interaction partner in the fully disordered state and folds while bound to the partner, i.e., folding is induced by the partner. In reality, one or the other process may occur, or also some combination of the two models. The full dimension of the structural flexibility of IDPs becomes obvious in cases, where one recognition domain of an IDP can adopt various structural folds upon interaction with different binding partners. One prominent example is the C-terminal disordered region of tumor suppressor p53, which can adopt helical, ß-strand or irregular structure upon interaction with different partners. In contrast to a concise disorder-to-order transition, some IDPs may also stay largely disordered and still show fast interconversion between coexisting conformations while in functional complex with a binding partner. Such heterogeneous protein complexes are referred to as 'fuzzy complexes'.

1.1.3 IDPs in diseases. Similarly to the formation of functional complexes with protein partners, a profound indentation in the free energy landscape of IDPs can be caused by formation of non-functional complexes of IDPs, like oligomers, amorphous aggregates and amyloid fibrils (Fig. 1B). Since IDPs are involved in many crucial cellular processes, e.g., signaling and regulation, misfolding of these proteins is often pathogenic. While some IDPs may exhibit an intrinsic propensity for forming a pathologic conformation, others are misfolding due to external factors, e.g., due to disrupted chaperone-interaction, point mutations, interaction with other proteins, toxins and small molecules or impaired post-translational modifications. The largest group of mis-folding diseases is characterized by the formation of stable, insoluble, highly organized filamentous protein aggregates known as amyloid fibrils, which accumulate in tissues or organs. Prominent among these amyloidogenic diseases are neurodegenerative diseases, where filamentous protein deposits are accumulating in the patient's brain. Striking examples are the IDPs a-synuclein, which is found as aggregated inclusions in the Lewy bodies in the brain of Parkinson's disease (PD) patients, and tau, which is a hallmark of Alzheimer's disease (AD) in its aggregated form.

1.2 Studying IDPs

Their prevalence in humans, their importance in a multitude of biological processes as well as their significance for health and disease makes IDPs a highly relevant and important subject of research. However, their structural and dynamical flexibility as well as the versatility of their shapes and appearances described above makes research on IDPs a challenging matter. Traditionally, protein structures are elucidated using NMR spectroscopy or X-ray crystallography. However, they are not straightforwardly applicable to obtain a conclusive analysis of the heterogeneous conformational ensemble that is characteristic for IDPs comprising a variety of quickly interconverting structures. Nonetheless, various methods of NMR spectroscopy, e.g., chemical shift dispersion, paramagnetic relaxation enhancement (PRE), and residual dipolar couplings (RDC), are widely applied for the investigation of IDPs. However, NMR techniques are subjected to limitations when it comes to investigation of large protein complexes, e.g., with lipids or other binding partners. Thus, investigation of IDPs in their relevant macromolecular context using NMR has its boundaries. Many other biophysical methods have been employed to investigate IDPs, among them circular dichroism (CD) spectroscopy, fluorescence resonance energy transfer (FRET), and many more. Since it is challenging to obtain meaningful experimental results on IDPs, computational approaches like prediction of intrinsic disorder or MD-simulations using experimental restraints, e.g., from PRE, are frequently used in IDP research. Commonly, the macroscopic morphology of IDPs in the fibril state is judged by transmission electron microscopy (TEM).

1.2.1 Electron paramagnetic resonance (EPR) for studying IDPs. A powerful experimental method that has become increasingly important in IDP research is electron paramagnetic resonance (EPR) spectroscopy. EPR is a versatile spectroscopy technique that allows us to study the dynamical and structural changes of proteins, in particular of IDPs. As it can only detect unpaired electrons, EPR spectroscopy is virtually background-free. EPR spectroscopy allows measurements of proteins of any size in arbitrarily complex environments, e.g., in the presence of binding partners like proteins, lipids or small molecules, as well as under arbitrary environmental conditions and even in the cell.

1.2.1.1 Site-directed spin labelling. EPR spectroscopy is sensitive to unpaired electrons only. Thus, in order to perform EPR measurements with many proteins, a paramagnetic center has to be introduced as a spin label via site-directed spin labelling (SDSL). Depending on the application, a suitable spin label has to be selected from a variety of possibilities. Some examples are shown in Fig. 2. For a recent review, the interested reader is referred to, e.g., Roser et al.

The spin labels most commonly used for EPR spectroscopy on proteins are nitroxide radicals. Among them, the methanethiosulfonate spin label (MTSL, see Fig. 2A), which is attached to a native or engineered cysteine residue in the protein via disulfide bonding, is most frequently used. However, in the reducing environment of a cell, both the N–O-moiety as well as the disulfide bond with the cysteine are susceptible to reduction. In order to ensure a stable attachment of the spin label to a cysteine, various linker-moieties have been developed, e.g., the maleimido moiety as used in 3-maleimido-proxyl (see Fig. 2B), which shows enhanced pH stability and is therefore often used for investigation of biological systems. For cysteine-specific attachment of a spin label, all native cysteine residues in the protein, which are not to be labelled, need to be replaced by non-reactive amino acids (often Ala, Ser) prior to labelling. A different and much more elegant way of introducing a spin label into a protein for in-cell EPR is via genetically encoded spin labelling, where the spin label is introduced as an unnatural amino acid with bio-orthogonal reactivity during the biosynthesis of the protein in vivo, making the technique especially suited for in-cell EPR. The corresponding unnatural amino acid SLK-1 is shown in Fig. 2C. When using this spin labelling approach, native cysteines present in the protein need not be substituted, implicating a more undisturbed protein than with cysteine-specific spin labelling. This also applies for a proposed spin labelling strategy based on click chemistry targeting unnatural amino acids. Approaches for stabilizing the nitroxide radical itself against reduction include the sterical shielding of the unpaired electron. A promising approach is the use of pyrrolidine-based nitroxides with ethyl groups slowing down the reduction process. As, in general, the stability of nitroxides against reduction is limited, other spin labels have been developed in recent years that pave the way towards in-cell EPR: Recently, a family of Gd3+-based spin labels has been introduced, which show superior stability in the cellular environment and exhibit a high sensitivity for EPR spectroscopy. Successful in cellula EPR distance measurements at Q- and W-band frequencies (34.5 and 95 GHz, respectively) have been reported with Gd3+-chelates like Gd3+-PyMTA (see Fig. 2D) or Gd3+-DOTA-M. Moreover, trityl radicals have been shown to be promising spin labels for in-cell EPR due to their favorable reduction characteristics. A different approach to spin labelling is the coordination of Cu2+-ions by two strategically placed histidine residues. EPR distances determined between two Cu2+-ions have been shown to be very narrow and readily relatable to the protein backbone structure.

In the investigation of spin-labelled protein oligomers, e.g., amyloid fibrils, several spin labels from various protein monomers might come into close contact. If contributions from inter-molecular spin–spin interactions are unwanted in the experiment, spin-labelled protein needs to be diluted with diamagnetic protein (Fig. 3).

1.2.1.2 Continuous wave (cw) EPR. Different methods of EPR spectroscopy allow accessing various types of information about the protein under investigation. There are two main experimental EPR techniques: On the one hand, in continuous-wave (cw) EPR spectroscopy the sample is subjected to continuous irradiation with microwave energy. Several excellent reviews describe the applications and developments in cw EPR for the investigation of proteins. Typical cw EPR experiments are performed at X-band microwave frequencies (9 GHz) and deliver information about the following sample parameters: (i) the mobility of the spin label side chain, (ii) the polarity of the spin label microenvironment, (iii) the solvent accessibility of the spin label side chain and (iv) distances to other paramagnetic centers. In the corresponding cw EPR experiments a site scan, i.e., performing the EPR experiment with several samples, where the spin label is attached to different positions of interest in the protein, helps to create a comprehensive picture of a protein region.

For the determination of the spin label mobility (i), the cw EPR experiment is performed at ambient temperature, or particularly at a physiologically relevant temperature. Most commonly, information about the reorientational dynamics of a spin label side chain is extracted from the EPR spectra via spectral shape analysis using full spectral simulations including the determination of the rotational correlation time tcorr using, e.g., the EasySpin software package. Cw EPR line shapes in X-band typically resolve spin label dynamics on a 100 ps to 100 ns timescale, which reflect side chain motions, backbone dynamics as well as tertiary contacts. Changes in the dynamics of a spin label side chain, e.g., due to interaction with a binding partner or conformational transitions of the protein can be detected with cw EPR spectroscopy: Even if the rate of conformational exchange is beyond the EPR timescale, the environment of the spin label is usually subjected to distinct changes, which allow the detection of distinct components in the EPR spectrum reflecting the different conformational states of the protein. Often, such EPR spectra contain not only one but several contributions from a superposition of various conformational states of the protein conformational ensemble. Multi-component spectral simulations allow to monitor and to quantify fractions of multiple structural states that coexist or exchange with each other.