Intrinsically Disordered Proteins and Their Environment: Effects of Strong Denaturants, Temperature, pH, Counter Ions, Membranes, Binding Partners, Osmolytes, and Macromolecular Crowding

Intrinsically disordered proteins (IDPs) differ from “normal” ordered proteins at several levels, structural, functional and conformational. Amino acid biases characteristic for IDPs determine their structural variability and lack of rigid well-folded structure. This structural plasticity is necessary for the unique functional repertoire of IDPs, which is complementary to the catalytic activities of ordered proteins. Amino acid biases also drive atypical responses of IDPs to changes in their environment. The conformational behavior of IDPs is characterized by the low cooperativity (or the complete lack thereof) of the denaturant-induced unfolding, lack of the measurable excess heat absorption peak(s) characteristic for the melting of ordered proteins, “turned out” response to heat and changes in pH, the ability to gain structure in the presence of various counter ions, osmolytes, membranes and binding partners, and by the unique response to macromolecular crowding. This review describes some of the most characteristic features of the IDP conformational behavior and the unique response of IDPs to changes in their environment.     

Mechanism and rate constants of the Cdc42 GTPase binding with intrinsically disordered effectors

Intrinsically disordered proteins (IDPs) are often involved in signaling and regulatory functions, through binding to cellular targets. Many IDPs undergo disorder‐to‐order transitions upon binding. Both the binding mechanisms and the magnitudes of the binding rate constants can have functional importance. Previously we have found that the coupled binding and folding of any IDP generally follows a sequential mechanism that we term dock‐and‐coalesce, whereby one segment of the IDP first docks to its subsite on the target surface and the remaining segments subsequently coalesce around their respective subsites. Here we applied our TransComp method within the framework of the dock‐and‐coalesce mechanism to dissect the binding kinetics of two Rho‐family GTPases, Cdc42 and TC10, with two intrinsically disordered effectors, WASP and Pak1. TransComp calculations identified the basic regions preceding the GTPase binding domains (GBDs) of the effectors as the docking segment. For Cdc42 binding with both WASP and Pak1, the calculated docking rate constants are close to the observed overall binding rate constants, suggesting that basic‐region docking is the rate‐limiting step and subsequent conformational coalescence of the GBDs on the Cdc42 surface is fast. The possibility that conformational coalescence of the WASP GBD on the TC10 surface is slow warrants further experimental investigation. The account for the differences in binding rate constants among the three GTPase‐effector systems and mutational effects therein yields deep physical and mechanistic insight into the binding processes. Our approach may guide the selection of mutations that lead to redesigned binding pathways. Proteins 2016; 84:674–685. © 2016 Wiley Periodicals, Inc.     

Intrinsically disordered proteins may escape unwanted interactions via functional misfolding

Intrinsically disordered proteins are highly abundant in nature and play a number of crucial roles in the living cells. They are commonly involved in a wide range of intermolecular interactions, and some of them possess remarkable binding promiscuity, being able to interact specifically with structurally unrelated partners. Although they do not have well-folded structure, some IDPs are known to fold at binding to their specific partners. IDPs are highly pliable and one IDP can form an array of unrelated structures being bound to different partners. It is believed that many IDPs, being mostly disordered, have transient elements of the preformed secondary structure which are highly interaction prone and is used by IDPs for binding to specific partners. The overall disordered nature of IDPs, their high conformational dynamics and flexibility, the presence of sticky preformed binding elements, and their ability to morph into differently-shaped bound configurations raised a very important question about the mechanisms preventing IDPs from unwanted interactions with non-native partners. In this review, a concept of functional misfolding is introduced. Accumulated to date data on the conformational behavior and fine structure of several IDPs suggest that the preformed binding elements might be involved in a set of non-native intramolecular interactions. In other words, there is a chance that a polypeptide chain misfolds to sequester the preformed elements inside the non-interactive or less-interactive cage, therefore preventing these elements from the unnecessary and unwanted interactions with non-native binding partners.     

NMR illuminates intrinsic disorder

Nuclear magnetic resonance (NMR) has long been instrumental in the characterization of intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs). This method continues to offer rich insights into the nature of IDPs in solution, especially in combination with other biophysical methods such as small-angle scattering, single-molecule fluorescence, electron paramagnetic resonance (EPR), and mass spectrometry. Substantial advances have been made in recent years in studies of proteins containing both ordered and disordered domains and in the characterization of problematic sequences containing repeated tracts of a single or a few amino acids. These sequences are relevant to disease states such as Alzheimer's, Parkinson's, and Huntington's diseases, where disordered proteins misfold into harmful amyloid. Innovative applications of NMR are providing novel insights into mechanisms of protein aggregation and the complexity of IDP interactions with their targets. As a basis for understanding the solution structural ensembles, dynamic behavior, and functional mechanisms of IDPs and IDRs, NMR continues to prove invaluable.     

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.     

Towards the physical basis of how intrinsic disorder mediates protein function

Intrinsically disordered proteins (IDPs) are an important class of functional proteins that is highly prevalent in biology and has broad association with human diseases. In contrast to structured proteins, free IDPs exist as heterogeneous and dynamical conformational ensembles under physiological conditions. Many concepts have been discussed on how such intrinsic disorder may provide crucial functional advantages, particularly in cellular signaling and regulation. Establishing the physical basis of these proposed phenomena requires not only detailed characterization of the disordered conformational ensembles, but also mechanistic understanding of the roles of various ensemble properties in IDP interaction and regulation. Here, we review the experimental and computational approaches that may be integrated to address many important challenges of establishing a "structural" basis of IDP function, and discuss some of the key emerging ideas on how the conformational ensembles of IDPs may mediate function, especially in coupled binding and folding interactions.     

CIDER: Resources to Analyze Sequence-Ensemble Relationships of Intrinsically Disordered Proteins

Intrinsically disordered proteins and regions (IDPs) represent a large class of proteins that are defined by conformational heterogeneity and lack of persistent tertiary/secondary structure. IDPs play important roles in a range of biological functions, and their dysregulation is central to numerous diseases, including neurodegeneration and cancer. The conformational ensembles of IDPs are encoded by their amino acid sequences. Here, we present two computational tools that are designed to enable rapid and high-throughput analyses of a wide range of physicochemical properties encoded by IDP sequences. The first, CIDER, is a user-friendly webserver that enables rapid analysis of IDP sequences. The second, localCIDER, is a high-performance software package that enables a wide range of analyses relevant to IDP sequences. In addition to introducing the two packages, we demonstrate the utility of these resources using examples where sequence analysis offers biophysical insights.     

Homogeneous and heterogeneous tertiary structure ensembles of amyloid-β peptides

The interplay of modern molecular simulation and high-quality nuclear magnetic resonance (NMR) experiments has reached a fruitful stage for quantitative characterization of structural ensembles of disordered peptides. Amyloid-β 1-42 (Aβ42), the primary peptide associated with Alzheimer's disease, and fragments such as Aβ21-30 are both classified as intrinsically disordered peptides (IDPs). We use a variety of NMR observables to validate de novo molecular dynamics simulations in explicit water to characterize the tertiary structure ensemble of Aβ42 and Aβ21-30 from the perspective of their classification as IDPs. Unlike the Aβ21-30 fragment that conforms to expectations of an IDP that is primarily extended, we find that Aβ42 samples conformations reflecting all possible secondary structure categories and spans the range of IDP classifications from collapsed structured states to highly extended conformations, making it an IDP with a far more heterogeneous tertiary ensemble.     

Not Only Expansion: Proline Content and Density Also Induce Disordered Protein Conformation Compaction

Intrinsically disordered proteins (IDPs) adopt a wide array of different conformations that can be constrained by the presence of proline residues, which are frequently found in IDPs. To assess the effects of proline, we designed a series of peptides that differ with respect to the number of prolines in the sequence and their organization. Using high-resolution atomistic molecular dynamics simulations, we found that accounting for whether the proline residues are clustered or isolated contributed significantly to explaining deviations in the experimentally-determined gyration radii of IDPs from the values expected based on the Flory scaling-law. By contrast, total proline content makes smaller contribution to explaining the effect of prolines on IDP conformation. Proline residues exhibit opposing effects depending on their organizational pattern in the IDP sequence. Clustered prolines (i.e., prolines with ≤2 intervening non-proline residues) result in expanded peptide conformations whereas isolated prolines (i.e., prolines with >2 intervening non-proline residues) impose compacted conformations. Clustered prolines were estimated to induce an expansion of ∼20% in IDP dimension (via formation of PPII structural elements) whereas isolated prolines were estimated to induce a compaction of ∼10% in IDP dimension (via the formation of backbone turns). This dual role of prolines provides a mechanism for conformational switching that does not rely on the kinetically much slower isomerization of cis proline to the trans form. Bioinformatic analysis demonstrates high populations of both isolated and clustered prolines and implementing them in coarse-grained molecular dynamics models illustrates that they improve the characterization of the conformational ensembles of IDPs.     

Intrinsically Disordered Protein Exhibits Both Compaction and Expansion under Macromolecular Crowding

Conformational malleability allows intrinsically disordered proteins (IDPs) to respond agilely to their environments, such as nonspecifically interacting with in vivo bystander macromolecules (or crowders). Previous studies have emphasized conformational compaction of IDPs due to steric repulsion by macromolecular crowders, but effects of soft attraction are largely unexplored. Here we studied the conformational ensembles of the IDP FlgM in both polymer and protein crowders by small-angle neutron scattering. As crowder concentrations increased, the mean radius of gyration of FlgM first decreased but then exhibited an uptick. Ensemble optimization modeling indicated that FlgM conformations under protein crowding segregated into two distinct populations, one compacted and one extended. Coarse-grained simulations showed that compacted conformers fit into an interstitial void and occasionally bind to a surrounding crowder, whereas extended conformers snake through interstitial crevices and bind multiple crowders simultaneously. Crowder-induced conformational segregation may facilitate various cellular functions of IDPs.     

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.     

How multisite phosphorylation impacts the conformations of intrinsically disordered proteins

Phosphorylation of intrinsically disordered proteins (IDPs) can produce changes in structural and dynamical properties and thereby mediate critical biological functions. How phosphorylation effects intrinsically disordered proteins has been studied for an increasing number of IDPs, but a systematic understanding is still lacking. Here, we compare the collapse propensity of four disordered proteins, Ash1, the C-terminal domain of RNA polymerase (CTD2’), the cytosolic domain of E-Cadherin, and a fragment of the p130Cas, in unphosphorylated and phosphorylated forms using extensive all-atom molecular dynamics (MD) simulations. We find all proteins to show V-shape changes in their collapse propensity upon multi-site phosphorylation according to their initial net charge: phosphorylation expands neutral or overall negatively charged IDPs and shrinks positively charged IDPs. However, force fields including those tailored towards and commonly used for IDPs overestimate these changes. We find quantitative agreement of MD results with SAXS and NMR data for Ash1 and CTD2’ only when attenuating protein electrostatic interactions by using a higher salt concentration (e.g. 350 mM), highlighting the overstabilization of salt bridges in current force fields. We show that phosphorylation of IDPs also has a strong impact on the solvation of the protein, a factor that in addition to the actual collapse or expansion of the IDP should be considered when analyzing SAXS data. Compared to the overall mild change in global IDP dimension, the exposure of active sites can change significantly upon phosphorylation, underlining the large susceptibility of IDP ensembles to regulation through post-translational modifications.     

Residual Structure Accelerates Binding of Intrinsically Disordered ACTR by Promoting Efficient Folding upon Encounter

Intrinsically disordered proteins (IDPs) often fold into stable structures upon specific binding. The roles of residual structure of unbound IDPs in coupling binding and folding have been under much debate. While many studies emphasize the importance of conformational flexibility for IDP recognition, it was recently demonstrated that stabilization the N-terminal helix of intrinsically disordered ACTR accelerated its binding to another IDP, NCBD of the CREB-binding protein. To understand how enhancing ACTR helicity accelerates binding, we derived a series of topology-based coarse-grained models that mimicked various ACTR mutants with increasing helical contents and reproduced their NCBD binding affinities. Molecular dynamics simulations were then performed to sample hundreds of reversible coupled binding and folding transitions. The results show that increasing ACTR helicity does not alter the baseline mechanism of synergistic folding, which continues to follow “extended conformational selection” with multiple stages of selection and induced folding. Importantly, these coarse-grained models, while only calibrated based on binding thermodynamics, recapitulate the observed kinetic acceleration with increasing ACTR helicity. However, the residual helices do not enhance the association kinetics via more efficient seeding of productive collisions. Instead, they allow the nonspecific collision complexes to evolve more efficiently into the final bound and folded state, which is the primary source of accelerated association kinetics. Meanwhile, reduced dissociation kinetics with increasing ACTR helicity can be directly attributed to smaller entropic cost of forming the bound state. Altogether, this study provides important mechanistic insights into how residual structure may modulate thermodynamics and kinetics of IDP interactions.     

Sequence Determinants of Compaction in Intrinsically Disordered Proteins

Intrinsically disordered proteins (IDPs), which lack folded structure and are disordered under nondenaturing conditions, have been shown to perform important functions in a large number of cellular processes. These proteins have interesting structural properties that deviate from the random-coil-like behavior exhibited by chemically denatured proteins. In particular, IDPs are often observed to exhibit significant compaction. In this study, we have analyzed the hydrodynamic radii of a number of IDPs to investigate the sequence determinants of this compaction. Net charge and proline content are observed to be strongly correlated with increased hydrodynamic radii, suggesting that these are the dominant contributors to compaction. Hydrophobicity and secondary structure, on the other hand, appear to have negligible effects on compaction, which implies that the determinants of structure in folded and intrinsically disordered proteins are profoundly different. Finally, we observe that polyhistidine tags seem to increase IDP compaction, which suggests that these tags have significant perturbing effects and thus should be removed before any structural characterizations of IDPs. Using the relationships observed in this analysis, we have developed a sequence-based predictor of hydrodynamic radius for IDPs that shows substantial improvement over a simple model based upon chain length alone.     

Uncoupled binding and folding of immune signaling-related intrinsically disordered proteins

The classical protein structure-function paradigm has been challenged by the emergence of intrinsically disordered proteins (IDPs), the proteins that do not adopt well-defined three-dimensional structures under physiological conditions. This development was accompanied by the introduction of a “coupled binding and folding” paradigm that suggests folding of IDPs upon binding to their partners. However, our recent studies challenge this general view by revealing a novel, previously unrecognized phenomenon – uncoupled binding and folding. This biologically important mechanism is characteristic of members of a new family of IDPs involved in immune signaling and underlies their unusual properties including: (1) specific homodimerization, (2) the lack of folding upon binding to a well-folded protein, another IDP molecule, or to lipid bilayer membranes, and (3) the “scissors-cut paradox”. The third phenomenon occurs in diverse IDP interactions and suggests that properties of IDP fragments are not necessarily additive in the context of the entire protein. The “no disorder-to-order transition” type of binding is distinct from known IDP interactions and is characterized by an unprecedented observation of the lack of chemical shift and peak intensity changes in multidimensional NMR spectra, a fingerprint of proteins, upon complex formation. Here, I focus on those interactions of IDPs with diverse biological partners where the binding phase driven by electrostatic interactions is not be necessarily followed by the hydrophobic folding phase. I also review new multidisciplinary knowledge about immune signaling-related IDPs and show how it expands our understanding of cell function with multiple applications in biology and medicine.     

Preserving free thiols of intrinsically disordered tau protein without the use of a reducing agent

Intrinsically disordered proteins (IDPs) represent key mediators in many physiological as well as pathological processes. Solution-exposed cysteines of IDPs are highly reactive and therefore reducing agents are frequently included during their preparation to prevent formation of nonnative disulfides. However, reductants can potentially interfere with subsequent assays performed on the purified IDPs. Herein we report a method for purification of IDP tau in an atmosphere of inert argon, which eliminates the need for reducing agents. We have used this method for preparing several IDP tau isoforms and found it useful in the investigation of monomeric tau toxicity in rat cerebral neurons.     

Effects of Molecular Crowding on the Dynamics of Intrinsically Disordered Proteins

Inside cells, the concentration of macromolecules can reach up to 400 g/L. In such crowded environments, proteins are expected to behave differently than in vitro. It has been shown that the stability and the folding rate of a globular protein can be altered by the excluded volume effect produced by a high density of macromolecules. However, macromolecular crowding effects on intrinsically disordered proteins (IDPs) are less explored. These proteins can be extremely dynamic and potentially sample a wide ensemble of conformations under non-denaturing conditions. The dynamic properties of IDPs are intimately related to the timescale of conformational exchange within the ensemble, which govern target recognition and how these proteins function. In this work, we investigated the macromolecular crowding effects on the dynamics of several IDPs by measuring the NMR spin relaxation parameters of three disordered proteins (ProTα, TC1, and α-synuclein) with different extents of residual structures. To aid the interpretation of experimental results, we also performed an MD simulation of ProTα. Based on the MD analysis, a simple model to correlate the observed changes in relaxation rates to the alteration in protein motions under crowding conditions was proposed. Our results show that 1) IDPs remain at least partially disordered despite the presence of high concentration of other macromolecules, 2) the crowded environment has differential effects on the conformational propensity of distinct regions of an IDP, which may lead to selective stabilization of certain target-binding motifs, and 3) the segmental motions of IDPs on the nanosecond timescale are retained under crowded conditions. These findings strongly suggest that IDPs function as dynamic structural ensembles in cellular environments.