Dynamical coupling of intrinsically disordered proteins and their hydration water: comparison with folded soluble and membrane proteins

Hydration water is vital for various macromolecular biological activities, such as specific ligand recognition, enzyme activity, response to receptor binding, and energy transduction. Without hydration water, proteins would not fold correctly and would lack the conformational flexibility that animates their three-dimensional structures. Motions in globular, soluble proteins are thought to be governed to a certain extent by hydration-water dynamics, yet it is not known whether this relationship holds true for other protein classes in general and whether, in turn, the structural nature of a protein also influences water motions. Here, we provide insight into the coupling between hydration-water dynamics and atomic motions in intrinsically disordered proteins (IDP), a largely unexplored class of proteins that, in contrast to folded proteins, lack a well-defined three-dimensional structure. We investigated the human IDP tau, which is involved in the pathogenic processes accompanying Alzheimer disease. Combining neutron scattering and protein perdeuteration, we found similar atomic mean-square displacements over a large temperature range for the tau protein and its hydration water, indicating intimate coupling between them. This is in contrast to the behavior of folded proteins of similar molecular weight, such as the globular, soluble maltose-binding protein and the membrane protein bacteriorhodopsin, which display moderate to weak coupling, respectively. The extracted mean square displacements also reveal a greater motional flexibility of IDP compared with globular, folded proteins and more restricted water motions on the IDP surface. The results provide evidence that protein and hydration-water motions mutually affect and shape each other, and that there is a gradient of coupling across different protein classes that may play a functional role in macromolecular activity in a cellular context.     

Meta-structure correlation in protein space unveils different selection rules for folded and intrinsically disordered proteins

The number of existing protein sequences spans a very small fraction of sequence space. Natural proteins have overcome a strong negative selective pressure to avoid the formation of insoluble aggregates. Stably folded globular proteins and intrinsically disordered proteins (IDPs) use alternative solutions to the aggregation problem. While in globular proteins folding minimizes the access to aggregation prone regions, IDPs on average display large exposed contact areas. Here, we introduce the concept of average meta-structure correlation maps to analyze sequence space. Using this novel conceptual view we show that representative ensembles of folded and ID proteins show distinct characteristics and respond differently to sequence randomization. By studying the way evolutionary constraints act on IDPs to disable a negative function (aggregation) we might gain insight into the mechanisms by which function-enabling information is encoded in IDPs.     

Composition-related structural transition of random peptides: insight into the boundary between intrinsically disordered proteins and folded proteins

Previous studies based on bioinformatics showed that there is a sharp distinction of structural features and residue composition between the intrinsically disordered proteins and the folded proteins. What induces such a composition-related structural transition? How do various kinds of interactions work in such processes? In this work, we investigate these problems based on a survey on peptides randomly composed of charged residues (including glutamic acids and lysines) and the residues with different hydrophobicity, such as alanines, glycines, or phenylalanines. Based on simulations using all-atom model and replica-exchange Monte Carlo method, a coil-globule transition is observed for each peptide. The corresponding transition temperature is found to be dependent on the contents of the hydrophobic and charged residues. For several cases, when the mean hydrophobicity is larger than a certain threshold, the transition temperature is higher than the room temperature, and vise versa. These thresholds of hydrophobicity and net charge are quantitatively consistent with the border line observed from the study of bioinformatics. These results outline the basic physical reasons for the compositional distinction between the intrinsically disordered proteins and the folded proteins. Furthermore, the contributions of various interactions to the structural variation of peptides are analyzed based on the contact statistics and the charge-pattern dependence of the gyration radii of the peptides. Our observations imply that the hydrophobicity contributes essentially to such composition-related transitions. Thus, we achieve a better understanding on composition–structure relation of the natural proteins and the underlying physics.     

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.     

Length-dependent compaction of intrinsically disordered proteins

This work investigates the effect of chain length on the degree of compaction of intrinsically disordered proteins (IDPs). The three main IDP types, native coil (NC), pre-molten globule (PMG) and molten globule (MG), are compared by means of a compaction index (CI) normalized for chain length. The results point out a strong variability of compactness as a function of chain length within each group, with larger proteins populating more compact states. While qualitative sequence features are responsible for the main differences among groups, chain length seems to have an unspecific effect modulating the extent of compaction within each group. The results are consistent with a cooperative character of the weak interactions responsible for chain collapse.     

Cold stability of intrinsically disordered proteins

Contrary to globular proteins, intrinsically disordered proteins (IDPs) lack a folded structure and they do not lose solubility at elevated temperatures. Although this should also be true at low temperatures, cold stability of IDPs has not been addressed in any scientific work so far. As direct characterization of cold-denaturation is difficult, we approached the problem through a freezing-induced loss-of-function model of globular-disordered functional protein pairs (m-calpain-calpastatin, tubulin-Map2c, Hsp90-ERD14). Our results affirm that in contrast with globular proteins IDPs are resistant to cold treatment. The theoretical and functional aspects of this observation are discussed.     

Constructing ensembles for intrinsically disordered proteins

The relatively flat energy landscapes associated with intrinsically disordered proteins makes modeling these systems especially problematic. A comprehensive model for these proteins requires one to build an ensemble consisting of a finite collection of structures, and their corresponding relative stabilities, which adequately capture the range of accessible states of the protein. In this regard, methods that use computational techniques to interpret experimental data in terms of such ensembles are an essential part of the modeling process. In this review, we critically assess the advantages and limitations of current techniques and discuss new methods for the validation of these ensembles.     

Binding cavities and druggability of intrinsically disordered proteins

To assess the potential of intrinsically disordered proteins (IDPs) as drug design targets, we have analyzed the ligand-binding cavities of two datasets of IDPs (containing 37 and 16 entries, respectively) and compared their properties with those of conventional ordered (folded) proteins. IDPs were predicted to possess more binding cavity than ordered proteins at similar length, supporting the proposed advantage of IDPs economizing genome and protein resources. The cavity number has a wide distribution within each conformation ensemble for IDPs. The geometries of the cavities of IDPs differ from the cavities of ordered proteins, for example, the cavities of IDPs have larger surface areas and volumes, and are more likely to be composed of a single segment. The druggability of the cavities was examined, and the average druggable probability is estimated to be 9% for IDPs, which is almost twice that for ordered proteins (5%). Some IDPs with druggable cavities that are associated with diseases are listed. The optimism versus obstacles for drug design for IDPs is also briefly discussed.     

Molecular dynamics simulation of intrinsically disordered proteins

Intrinsically disordered proteins are biomolecules that do not have a definite 3D structure; therefore, their dynamical simulation cannot start from a known list of atomistic positions, such as a Protein Data Bank file. We describe a method to start a computer simulation of these proteins. The first step of the procedure is the creation of a multi-rod configuration of the molecule, derived from its primary sequence. This structure is dynamically evolved in vacuo until its gyration radius reaches the experimental average value; at this point solvent molecules, in explicit or implicit implementation, are added to the protein and a regular molecular dynamics simulation follows. We have applied this procedure to the simulation of tau, one of the largest totally disordered proteins.     

Binding of intrinsically disordered proteins is not necessarily accompanied by a structural transition to a folded form

There are a large number of protein domains and even entire proteins, lacking ordered structure under physiological conditions. Intriguingly, a highly flexible, random coil-like conformation is the native and functional state for many proteins known to be involved in cell signaling. An example is a key component of immune signaling, the cytoplasmic region of the T cell receptor zeta subunit. This domain exhibits specific dimerization that is distinct from non-specific aggregation behavior seen in many systems. In this work, we use diffusion and chemical shift mapping NMR data to show that the protein does not undergo a transition between disordered and ordered states upon dimerization. This finding opposes the generally accepted view on the behavior of intrinsically disordered proteins, provides evidence for the existence of specific dimerization interactions for intrinsically disordered protein species and opens a new line of research in this new and quickly developing field.     

Residue packing in globular and intrinsically disordered proteins

Intrinsically disordered proteins (IDPs)/regions do not have well‐defined secondary and tertiary structures, however, they are functional and it is critical to gain a deep understanding of their residue packing. The shape distributions methodology, which is usually utilized in pattern recognition, clustering, and classification studies in computer science, may be adopted to study the residue packing of the proteins. In this study, shape distributions of the globular proteins and IDPs were obtained to shed light on the residue packing of their structures. The shape feature that was used is the sphericity of tetrahedra obtained by Delaunay Tessellation of points of Cα coordinates. Then the sphericity probability distributions were compared by using Principal Component Analysis. This computational structural study shows that the set of IDPs constitute a more diverse set than the set of globular proteins in terms of the geometrical properties of their network structures.     

Protein kinases phosphorylate long disordered regions in intrinsically disordered proteins

Phosphorylation is a major post‐translational modification that plays a central role in signaling pathways. Protein kinases phosphorylate substrates (phosphoproteins) by adding phosphate at Ser/Thr or Tyr residues (phosphosites). A large amount of data identifying and describing phosphosites in phosphoproteins has been reported but the specificity of phosphorylation is not fully resolved. In this report, data of kinase‐substrate pairs identified by the Kinase‐Interacting Substrate Screening (KISS) method were used to analyze phosphosites in intrinsically disordered regions (IDRs) of intrinsically disordered proteins. We compared phosphorylated and nonphosphorylated IDRs and found that the phosphorylated IDRs were significantly longer than nonphosphorylated IDRs. The phosphorylated IDR is often the longest IDR (71%) in a phosphoprotein when only a single phosphosite exists in the IDR, and when the phosphoprotein has multiple phosphosites in an IDR(s), the phosphosites are primarily localized in a single IDR (78%) and this IDR is usually the longest one (81%). We constructed a stochastic model of phosphorylation to estimate the effect of IDR length. The model that accounted for IDR length produced more realistic results when compared with a model that excluded the IDR length. We propose that the IDR length is a significant determinant for locating kinase phosphorylation sites in phosphoproteins.     

Context-dependent resistance to proteolysis of intrinsically disordered proteins

Intrinsically disordered proteins (IDPs), also known as intrinsically unstructured proteins (IUPs), lack a well-defined 3D structure in vitro and, in some cases, also in vivo. Here, we discuss the question of proteolytic sensitivity of IDPs, with a view to better explaining their in vivo characteristics. After an initial assessment of the status of IDPs in vivo, we briefly survey the intracellular proteolytic systems. Subsequently, we discuss the evidence for IDPs being inherently sensitive to proteolysis. Such sensitivity would not, however, result in enhanced degradation if the protease-sensitive sites were sequestered. Accordingly, IDP access to and degradation by the proteasome, the major proteolytic complex within eukaryotic cells, are discussed in detail. The emerging picture appears to be that IDPs are inherently sensitive to proteasomal degradation along the lines of the "degradation by default" model. However, available data sets of intracellular protein half-lives suggest that intrinsic disorder does not imply a significantly shorter half-life. We assess the power of available systemic half-life measurements, but also discuss possible mechanisms that could protect IDPs from intracellular degradation. Finally, we discuss the relevance of the proteolytic sensitivity of IDPs to their function and evolution.     

Prediction of short loops in intrinsically disordered proteins

A new possibility of predicting short disordered regions (loops) at a small window size (three amino acid residues) by the FoldUnfold program is described. As demonstrated with the example of three G proteins, FoldUnfold predicted almost all existing loops at the positions fitting well the X-ray structural data. The loops predicted in the Ras p21 structure were classified into two types. The loops of the first type display high Debye-Waller factor values, characteristic of the so-called functional loops (flexible loops). The second-type loops had lower Debye-Waller factor values and, consequently, were regarded as the loops connecting secondary structure elements (rigid loops). Comparison of the results predicted by FoldUnfold with the predictions of other programs (PONDR, RONN, DisEMBL, PreLINK, IUPred, GlobPlot 2, and FoldIndex) demonstrated that the first program was much better in predicting the positions of short loops. FoldUnfold made it possible to solve the problem difficult for the other programs, that is, to determine the boundary between the ordered and disordered regions in proteins with a large fraction of disordered regions, exemplified by the ubiquitin-like domain. In particular, FoldUnfold predicted a boundary between the ordered and disordered regions at residues 30 and 31, whereas the other programs predicted the boundary in the range of 28–70 amino acid residues.     

Expanding the proteome: disordered and alternatively folded proteins

Proteins provide much of the scaffolding for life, as well as undertaking a variety of essential catalytic reactions. These characteristic functions have led us to presuppose that proteins are in general functional only when well structured and correctly folded. As we begin to explore the repertoire of possible protein sequences inherent in the human and other genomes, two stark facts that belie this supposition become clear: firstly, the number of apparent open reading frames in the human genome is significantly smaller than appears to be necessary to code for all of the diverse proteins in higher organisms, and secondly that a significant proportion of the protein sequences that would be coded by the genome would not be expected to form stable three-dimensional (3D) structures. Clearly the genome must include coding for a multitude of alternative forms of proteins, some of which may be partly or fully disordered or incompletely structured in their functional states. At the same time as this likelihood was recognized, experimental studies also began to uncover examples of important protein molecules and domains that were incompletely structured or completely disordered in solution, yet remained perfectly functional. In the ensuing years, we have seen an explosion of experimental and genome-annotation studies that have mapped the extent of the intrinsic disorder phenomenon and explored the possible biological rationales for its widespread occurrence. Answers to the question 'why would a particular domain need to be unstructured?' are as varied as the systems where such domains are found. This review provides a survey of recent new directions in this field, and includes an evaluation of the role not only of intrinsically disordered proteins but also of partially structured and highly dynamic members of the disorder-order continuum.