Unusual biophysics of intrinsically disordered proteins

Research of a past decade and a half leaves no doubt that complete understanding of protein functionality requires close consideration of the fact that many functional proteins do not have well-folded structures. These intrinsically disordered proteins (IDPs) and proteins with intrinsically disordered protein regions (IDPRs) are highly abundant in nature and play a number of crucial roles in a living cell. Their functions, which are typically associated with a wide range of intermolecular interactions where IDPs possess remarkable binding promiscuity, complement functional repertoire of ordered proteins. All this requires a close attention to the peculiarities of biophysics of these proteins. In this review, some key biophysical features of IDPs are covered. In addition to the peculiar sequence characteristics of IDPs these biophysical features include sequential, structural, and spatiotemporal heterogeneity of IDPs; their rough and relatively flat energy landscapes; their ability to undergo both induced folding and induced unfolding; the ability to interact specifically with structurally unrelated partners; the ability to gain different structures at binding to different partners; and the ability to keep essential amount of disorder even in the bound form. IDPs are also characterized by the “turned-out” response to the changes in their environment, where they gain some structure under conditions resulting in denaturation or even unfolding of ordered proteins. It is proposed that the heterogeneous spatiotemporal structure of IDPs/IDPRs can be described as a set of foldons, inducible foldons, semi-foldons, non-foldons, and unfoldons. They may lose their function when folded, and activation of some IDPs is associated with the awaking of the dormant disorder. It is possible that IDPs represent the “edge of chaos” systems which operate in a region between order and complete randomness or chaos, where the complexity is maximal. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.     

A decade and a half of protein intrinsic disorder: Biology still waits for physics

The abundant existence of proteins and regions that possess specific functions without being uniquely folded into unique 3D structures has become accepted by a significant number of protein scientists. Sequences of these intrinsically disordered proteins (IDPs) and IDP regions (IDPRs) are characterized by a number of specific features, such as low overall hydrophobicity and high net charge which makes these proteins predictable. IDPs/IDPRs possess large hydrodynamic volumes, low contents of ordered secondary structure, and are characterized by high structural heterogeneity. They are very flexible, but some may undergo disorder to order transitions in the presence of natural ligands. The degree of these structural rearrangements varies over a very wide range. IDPs/IDPRs are tightly controlled under the normal conditions and have numerous specific functions that complement functions of ordered proteins and domains. When lacking proper control, they have multiple roles in pathogenesis of various human diseases. Gaining structural and functional information about these proteins is a challenge, since they do not typically “freeze” while their “pictures are taken.” However, despite or perhaps because of the experimental challenges, these fuzzy objects with fuzzy structures and fuzzy functions are among the most interesting targets for modern protein research. This review briefly summarizes some of the recent advances in this exciting field and considers some of the basic lessons learned from the analysis of physics, chemistry, and biology of IDPs.     

Criticality in the conformational phase transition among self-similar groups in intrinsically disordered proteins: Probed by salt-bridge dynamics

Intrinsically disordered proteins (IDP) serve as one of the key components in the global proteome. In contrast to globular proteins, they harbor an enormous amount of physical flexibility enforcing them to be retained in conformational ensembles rather than stable folds. Previous studies in an aligned direction have revealed the importance of transient dynamical phenomena like that of salt-bridge formation in IDPs to support their physical flexibility and have further highlighted their functional relevance. For this characteristic flexibility, IDPs remain amenable and accessible to different ordered binding partners, supporting their potential multi-functionality. The current study further addresses this complex structure-functional interplay in IDPs using phase transition dynamics to conceptualize the underlying (avalanche type) mechanism of their being distributed across and hopping around degenerate structural states (conformational ensembles). For this purpose, extensive molecular dynamics simulations have been done and the data analyzed from a statistical physics perspective. Investigation of the plausible scope of 'self-organized criticality' (SOC) to fit into the complex dynamics of IDPs was found to be assertive, relating the conformational degeneracy of these proteins to their functional multiplicity. In accordance with the transient nature of 'salt-bridge dynamics', the study further uses it as a probe to explain the structural basis of the proposed criticality in the conformational phase transition among self-similar groups in IDPs. The analysis reveal scale-invariant self-similar fractal geometries in the structural conformations of different IDPs. The insights from the study has the potential to be extended further to benefit structural tinkering of IDPs in their functional characterization and drugging.     

Do sequence neighbours of intrinsically disordered regions promote structural flexibility in intrinsically disordered proteins?

Intrinsically disordered proteins (IDPs) are crucial players in various cellular activities. Several experimental and computational analyses have been conducted to study structural pliability and functional potential of IDPs. In spite of active research in past few decades, what induces structural disorder in IDPs and how is still elusive. Many studies testify that sequential and spatial neighbours often play important roles in determining structural and functional behaviour of proteins. Considering this fact, we assessed sequence neighbours of intrinsically disordered regions (IDRs) to understand if they have any role to play in inducing structural flexibility in IDPs. Our analysis includes 97% eukaryotic IDPs and 3% from bacteria and viruses. Physicochemical and structural parameters including amino acid propensity, hydrophobicity, secondary structure propensity, relative solvent accessibility, B-factor and atomic packing density are used to characterise the neighbouring residues of IDRs (NRIs). We show that NRIs exhibit a unique nature, which makes them stand out from both ordered and disordered residues. They show correlative occurrences of residue pairs like Ser-Thr and Gln-Asn, indicating their tendency to avoid strong biases of order or disorder promoting amino acids. We also find differential preferences of amino acids between N- and C-terminal neighbours, which might indicate a plausible directional effect on the dynamics of adjacent IDRs. We designed an efficient prediction tool using Random Forest to distinguish the NRIs from the ordered residues. Our findings will contribute to understand the behaviour of IDPs, and may provide potential lead in deciphering the role of IDRs in protein folding and assembly.     

On the link between conformational changes,ligand binding and heat capacity

BackgroundConformational changes coupled to ligand binding constitute the structural and energetics basis underlying cooperativity, allostery and, in general, protein regulation. These conformational rearrangements are associated with heat capacity changes. ITC is a unique technique for studying binding interactions because of the simultaneous determination of the binding affinity and enthalpy, and for providing the best estimates of binding heat capacity changes.Scope of reviewStill controversial issues in ligand binding are the discrimination between the “conformational selection model” and the “induced fit model”, and whether or not conformational changes lead to temperature dependent apparent binding heat capacities. The assessment of conformational changes associated with ligand binding by ITC is discussed. In addition, the “conformational selection” and “induced fit” models are reconciled, and discussed within the context of intrinsically (partially) unstructured proteins.Major conclusionsConformational equilibrium is a major contribution to binding heat capacity changes. A simple model may explain both conformational selection and induced fit scenarios. A temperature-independent binding heat capacity does not necessarily indicate absence of conformational changes upon ligand binding. ITC provides information on the energetics of conformational changes associated with ligand binding (and other possible additional coupled equilibria).General significancePreferential ligand binding to certain protein states leads to an equilibrium shift that is reflected in the coupling between ligand binding and additional equilibria. This represents the structural/energetic basis of the widespread dependence of ligand binding parameters on temperature, as well as pH, ionic strength and the concentration of other chemical species. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Modulation of Correlated Segment Fluctuations in IDPs upon Complex Formation as an Allosteric Regulatory Mechanism

Molecular recognition of and by intrinsically disordered proteins (IDPs) is an intriguing and still largely elusive phenomenon. Typically, protein recognition involving IDPs requires either folding upon binding or, alternatively, the formation of “fuzzy complexes.” Here we show via correlation analyses of paramagnetic relaxation enhancement data unprecedented and striking alterations of the concerted fluctuations within the conformational ensemble of IDPs upon ligand binding. We study the binding of α-synuclein to calmodulin, a ubiquitous calcium-binding protein, and the binding of the extracellular matrix IDP osteopontin to heparin, a mimic of the extracellular matrix ligand hyaluronic acid. In both cases, binding leads to reduction of correlated long-range motions in these two IDPs and thus indicates a loosening of structural compaction upon binding. Most importantly, however, the simultaneous presence of correlated and anti-correlated fluctuations in IDPs suggests the prevalence of “energetic frustration” and provides an explanation for the puzzling observation of disordered allostery in IDPs.     

Targeting intrinsically disordered proteins in neurodegenerative and protein dysfunction diseases: another illustration of the D2 concept

Many biologically active proteins, which are usually called intrinsically disordered or natively unfolded proteins, lack stable tertiary and/or secondary structure under physiological conditions in vitro. Their functions complement the functional repertoire of ordered proteins, with intrinsically disordered proteins (IDPs) often being involved in regulation, signaling and control. Their amino acid sequences and compositions are very different from those of ordered proteins, making reliable identification of IDPs possible at the proteome level. IDPs are highly abundant in various human diseases, including neurodegeneration and other protein dysfunction maladies and, therefore, represent attractive novel drug targets. Some of the aspects of IDPs, as well as their roles in neurodegeneration and protein dysfunction diseases, are discussed in this article, together with the peculiarities of IDPs as potential drug targets.     

Structural changes and fluctuations of proteins: I. A statistical thermodynamic model

A general theory of the structural changes and fluctuations of proteins has been proposed based on statistical thermodynanic considerations at the chain level.The “structure” of protein was assumed to be characterized by the state of secondary bonds between unique pairs of specific sites on peptide chains. Every secondary bond changes between the bonded and unboned states by thermal agitation and the “structure” is continuously fluctuating. The free energy of the “structural state” that is defined by the fraction of secondary bonds in the bonded state has been expressed by the bond energy, the cooperative interaction between bonds, the mixing entropy of bonds, and the entropy of polypeptide chains. The most probable “structural state” can be simply determined by graphical analysis and the effect of temperature or solvent composition on it is discussed. The temperature dependence of the free energy, the probability distribution of structural states and the specific heat have been calculated for two examples of structural change.The theory predicts two different types of structural changes from the ordered to disordered state, a “structural transition” and a “gradual structural change” with rising temperature, In the “structural transition”, the probability distribution has two maxima in the temperature range of transition. In the “gradual structural change”, the probability distribution has only one maximum during the change.A considerable fraction of secondary bonds is in the unbonded state and is always fluctuating even in the ordered state at room temperature. Such structural fluctuations in a single protein molecule have been discussed quantitatively.The theory is extended to include small molecules which bind to the protein molecule and affect the structural state. The changes of structural state caused by specific and non-specific binding and allosteric effects are explained in a unified manner.     

Unveiling induced folding of intrinsically disordered proteins – Protein engineering,frustration and emerging themes

Intrinsically disordered proteins (IDPs) can be generally described as a class of proteins that lack a well-defined ordered structure in isolation at physiological conditions. Upon binding to their physiological ligands, IDPs typically undergo a disorder-to-order transition, which may or may not lead to the complete folding of the IDP. In this short review, we focus on some of the key findings pertaining to the mechanisms of such induced folding. In particular, first we describe the general features of the reaction; then, we discuss some of the most remarkable findings obtained from applying protein engineering in synergy with kinetic studies to induced folding; and finally, we offer a critical view on some of the emerging themes when considering the structural heterogeneity of IDPs vis-à-vis to their inherent frustration.     

What does it mean to be natively unfolded?

Natively unfolded or intrinsically unstructured proteins constitute a unique group of the protein kingdom. The evolutionary persistence of such proteins represents strong evidence in the favor of their importance and raises intriguing questions about the role of protein disorders in biological processes. Additionally, natively unfolded proteins, with their lack of ordered structure, represent attractive targets for the biophysical studies of the unfolded polypeptide chain under physiological conditions in vitro. The goal of this study was to summarize the structural information on natively unfolded proteins in order to evaluate their major conformational characteristics. It appeared that natively unfolded proteins are characterized by low overall hydrophobicity and large net charge. They possess hydrodynamic properties typical of random coils in poor solvent, or premolten globule conformation. These proteins show a low level of ordered secondary structure and no tightly packed core. They are very flexible, but may adopt relatively rigid conformations in the presence of natural ligands. Finally, in comparison with the globular proteins, natively unfolded polypeptides possess 'turn out' responses to changes in the environment, as their structural complexities increase at high temperature or at extreme pH.     

Features of molecular recognition of intrinsically disordered proteins via coupled folding and binding

The sequence–structure–function paradigm of proteins has been revolutionized by the discovery of intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs). In contrast to traditional ordered proteins, IDPs/IDRs are unstructured under physiological conditions. The absence of well‐defined three‐dimensional structures in the free state of IDPs/IDRs is fundamental to their function. Folding upon binding is an important mode of molecular recognition for IDPs/IDRs. While great efforts have been devoted to investigating the complex structures and binding kinetics and affinities, our knowledge on the binding mechanisms of IDPs/IDRs remains very limited. Here, we review recent advances on the binding mechanisms of IDPs/IDRs. The structures and kinetic parameters of IDPs/IDRs can vary greatly, and the binding mechanisms can be highly dependent on the structural properties of IDPs/IDRs. IDPs/IDRs can employ various combinations of conformational selection and induced fit in a binding process, which can be templated by the target and/or encoded by the IDP/IDR. Further studies should provide deeper insights into the molecular recognition of IDPs/IDRs and enable the rational design of IDP/IDR binding mechanisms in the future.     

A J-modulated protonless NMR experiment characterizes the conformational ensemble of the intrinsically disordered protein WIP

Intrinsically disordered proteins (IDPs) are multi-conformational polypeptides that lack a single stable three-dimensional structure. It has become increasingly clear that the versatile IDPs play key roles in a multitude of biological processes, and, given their flexible nature, NMR is a leading method to investigate IDP behavior on the molecular level. Here we present an IDP-tailored J-modulated experiment designed to monitor changes in the conformational ensemble characteristic of IDPs by accurately measuring backbone one- and two-bond J(¹⁵N,¹³Cα) couplings. This concept was realized using a unidirectional (H)NCO ¹³C-detected experiment suitable for poor spectral dispersion and optimized for maximum coverage of amino acid types. To demonstrate the utility of this approach we applied it to the disordered actin-binding N-terminal domain of WASp interacting protein (WIP), a ubiquitous key modulator of cytoskeletal changes in a range of biological systems. One- and two-bond J(¹⁵N,¹³Cα) couplings were acquired for WIP residues 2–65 at various temperatures, and in denaturing and crowding environments. Under native conditions fitted J-couplings identified in the WIP conformational ensemble a propensity for extended conformation at residues 16–23 and 45–60, and a helical tendency at residues 28–42. These findings are consistent with a previous study of the based upon chemical shift and RDC data and confirm that the WIP^2–65 conformational ensemble is biased towards the structure assumed by this fragment in its actin-bound form. The effects of environmental changes upon this ensemble were readily apparent in the J-coupling data, which reflected a significant decrease in structural propensity at higher temperatures, in the presence of 8 M urea, and under the influence of a bacterial cell lysate. The latter suggests that crowding can cause protein unfolding through protein–protein interactions that stabilize the unfolded state. We conclude that J-couplings are a useful measureable in characterizing structural ensembles in IDPs, and that the proposed experiment provides a practical method for accurately performing such measurements, once again emphasizing the power of NMR in studying IDP behavior.     

Temperature-dependent structural changes in intrinsically disordered proteins: Formation of α-helices or loss of polyproline II?

Structural characterization of intrinsically disordered proteins (IDPs) is mandatory for deciphering their potential unique physical and biological properties. A large number of circular dichroism (CD) studies have demonstrated that a structural change takes place in IDPs with increasing temperature, which most likely reflects formation of transient α-helices or loss of polyproline II (PPII) content. Using three IDPs, ACTR, NHE1, and Spd1, we show that the temperature-induced structural change is common among IDPs and is accompanied by a contraction of the conformational ensemble. This phenomenon was explored at residue resolution by multidimensional NMR spectroscopy. Intrinsic chemical shift referencing allowed us to identify regions of transiently formed helices and their temperature-dependent changes in helicity. All helical regions were found to lose rather than gain helical structures with increasing temperature, and accordingly these were not responsible for the change in the CD spectra. In contrast, the nonhelical regions exhibited a general temperature-dependent structural change that was independent of long-range interactions. The temperature-dependent CD spectroscopic signature of IDPs that has been amply documented can be rationalized to represent redistribution of the statistical coil involving a general loss of PPII conformations.     

Self-assembling systems comprising intrinsically disordered protein polymers like elastin-like recombinamers

Despite lacking cooperatively folded structures under native conditions, numerous intrinsically disordered proteins (IDPs) nevertheless have great functional importance. These IDPs are hybrids containing both ordered and intrinsically disordered protein regions (IDPRs), the structure of which is highly flexible in this unfolded state. The conformational flexibility of these disordered systems favors transitions between disordered and ordered states triggered by intrinsic and extrinsic factors, folding into different dynamic molecular assemblies to enable proper protein functions. Indeed, prokaryotic enzymes present less disorder than eukaryotic enzymes, thus showing that this disorder is related to functional and structural complexity. Protein-based polymers that mimic these IDPs include the so-called elastin-like polypeptides (ELPs), which are inspired by the composition of natural elastin. Elastin-like recombinamers (ELRs) are ELPs produced using recombinant techniques and which can therefore be tailored for a specific application. One of the most widely used and studied characteristic structures in this field is the pentapeptide (VPGXG)_n. The structural disorder in ELRs probably arises due to the high content of proline and glycine in the ELR backbone, because both these amino acids help to keep the polypeptide structure of elastomers disordered and hydrated. Moreover, the recombinant nature of these systems means that different sequences can be designed, including bioactive domains, to obtain specific structures for each application. Some of these structures, along with their applications as IDPs that self-assemble into functional vesicles or micelles from diblock copolymer ELRs, will be studied in the following sections. The incorporation of additional order- and disorder-promoting peptide/protein domains, such as α-helical coils or β-strands, in the ELR sequence, and their influence on self-assembly, will also be reviewed. In addition, chemically cross-linked systems with controllable order–disorder balance, and their role in biomineralization, will be discussed. Finally, we will review different multivalent IDPs-based coatings and films for different biomedical applications, such as spatially controlled cell adhesion, osseointegration, or biomaterial-associated infection (BAI).     

Application of native mass spectrometry in studying intrinsically disordered proteins: A special focus on neurodegenerative diseases

Intrinsically disordered proteins (IDPs) are integral part of the proteome, regulating vital biological processes. Such proteins gained further visibility due to their key role in neurodegenerative diseases and cancer. IDPs however, escape structural characterization by traditional biophysical tools owing to their extreme flexibility and heterogeneity. In this review, we discuss the advantages of native mass spectrometry (MS) in analysing the atypical conformational dynamics of IDPs and recent advances made in the field. Especially, MS studies unravelling the conformational facets of IDPs involved in neurodegenerative diseases are highlighted. The limitations and the future promises of native MS while studying IDPs have been discussed.     

Monitoring the conformational changes of an intrinsically disordered peptide using a quartz crystal microbalance

Intrinsically disordered peptides (IDPs) have recently garnered much interest because of their role in biological processes such as molecular recognition and their ability to undergo stimulus-responsive conformational changes. The block V repeat-in-toxin motif of the Bordetella pertussis adenylate cyclase is an example of an IDP that undergoes a transition from a disordered state to an ordered beta roll conformation in the presence of calcium ions. In solution, a C-terminal capping domain is necessary for this transition to occur. To further explore the conformational behavior and folding requirements of this IDP, we have cysteine modified three previously characterized constructs, allowing for attachment to the gold surface of a quartz crystal microbalance (QCM). We demonstrate that, while immobilized, the C-terminally capped peptide exhibits similar calcium-binding properties to what have been observed in solution. In addition, immobilization on the solid surface appears to enable calcium-responsiveness in the uncapped peptides, in contrast to the behavior observed in solution. This work demonstrates the power of QCM as a tool to study the conformational changes of IDPs immobilized on surfaces and has implications for a range of potential applications where IDPs may be engineered and used including protein purification, biosensors, and other bionanotechnology applications.