Most cytosolic eukaryotic proteins contain a mixture of ordered and disordered regions. Disordered regions facilitate cell signaling by concentrating sites for posttranslational modifications and protein–protein interactions into arrays of short linear motifs that can be reorganized by RNA splicing. The evolution of disordered regions looks different from their ordered counterparts. In some cases, selection is focused on maintaining protein binding interfaces and PTM sites, but sequence heterogeneity is common. In other cases, simple properties like charge, length, or end-to-end distance are maintained. Many disordered protein binding sites contain some transient secondary structure that may resemble the structure of the bound state. α-Helical secondary structure is common and a wide range of fractional helicity is observed in different disordered regions. Here we provide a simple protocol to identify transient helical segments and design mutants that can change their structure and function.
Within the arsenal of experimental methods in studying intrinsically disordered proteins (IDPs), single-molecule fluorescence methods provide unique long-range distance and dynamics information. In this chapter, we discuss recent progress on using single-molecule fluorescence methods for probing both intra- and intermolecular interactions of IDPs. We focus on several aspects, including models for analyzing experimental data, physical principles that can be obtained, and combinations with other experimental methods for a comprehensive view of the structural properties of IDPs.
Nuclear magnetic resonance is a unique approach to obtain information on dynamics over a broad range of timescales and high-resolution information in disordered proteins. In this chapter, we will review the different methods available to characterize motions in disordered proteins. We first focus on dynamics in the picosecond-nanosecond range, which is characterized by nuclear spin relaxation measurements. We will focus then on slower processes, mostly in the millisecond range, studied by chemical-exchange NMR. We will discuss in particular the dynamics of the binding of disordered proteins to their targets and their dynamics in these complexes.
Over the last decade, there has been considerable interest in the role of intrinsically disordered proteins and protein regions in the formation of nonstoichiometric assemblies. These assemblies have been given various names, including membraneless organelles and biomolecular condensates, and their formation is often described as a biological phase transition. Given that some of these assemblies are highly dynamic and undergo dripping, wetting, and fusion, the physics of liquid-liquid phase separation have been invoked as a putative mechanism for assembly formation and maintenance. In this chapter, we first introduce some general background on biological phase separation and then consider how IDRs may mediate this phenomenon. We will focus on in vitro systems for which sequence effects have been explicitly examined while considering the caveats and limitations of projecting in vitro observations to in vivo phenomena. We finish by considering some additional biophysical and functional topics that have been discussed less explicitly elsewhere.
Intrinsically disordered proteins (IDPs), polypeptide chains that do not fold into a homogeneous three-dimensional (3D) structure, are overrepresented in major human disease pathways. As such, IDPs hold tremendous potential for therapeutic intervention. To date, more than a dozen IDPs have been targeted and several inhibitors have reached clinical trials, demonstrating that IDPs are indeed druggable. Despite these advances, no IDP-targeting compound has made it onto the market, which calls for the use of novel concepts and approaches in order to turn IDP inhibitors into commercial drugs. The molecular mechanisms of action of current drug-like candidates are manifold: (i) inhibition of protein-protein interactions, (ii) direct binding to monomeric IDPs shifting their conformational ensembles, (iii) distortion of their functional complexes, and (iv) disassembly of intermediate oligomeric species critical for amyloid fibril formation. In the following, we review computational and experimental technologies that have been successfully applied to screen and characterize modulators of IDP function, and present prospects for the future of drug discovery targeting IDPs.
The function of intrinsically disordered proteins and regions has to be described in terms of ensembles of structural conformations that allow one to predict observables for comparison with experimental values. Herein we describe how to derive such structural ensembles from a combination of experimental evidence and computational techniques. We compare different approaches and review typical applications of ensemble descriptions in structural biology.
Intrinsically disordered proteins (IDPs) are strongly represented in functional roles that involve binding interactions with other proteins, such as signaling and regulation. Experimental studies of IDP binding, while challenging, have in recent years increasingly enhanced our understanding of the principles and mechanisms that IDPs employ to interact with their partners. We give an overview of experimental techniques that can be used to study IDP binding, and we attempt to classify experimentally characterized IDP interactions according to three distinctive parameters: level of structure formation and dynamics in the complex, length of the interacting segment(s), and valency. We then review the experimental literature with regard to properties that have been described as characteristic for IDP binding, such as affinity and specificity, as well as association and dissociation rates. We also comment on mechanistic questions such as whether IDP binding is mediated more by conformational selection or by induced fit, and whether folding upon binding of an IDP is encoded in its sequence or templated by the partner.
Intrinsically disordered proteins (IDPs) are abundant in proteomes, especially in eukaryotes. Protein-protein interactions (PPIs) involving an IDP are major players in the PPI network, comprising an estimated 15%–45% of all interactions. These disordered PPIs are prevalent in various important cellular processes and are associated with allosteric regulation, posttranslational modification, and alternative splicing. While IDPs lack a stable tertiary structure in isolation, they often become ordered when binding to a stable partner. The tertiary structure of the complex provides a molecular basis for understanding those pathways and related phenomena. Because capturing the IDP structures through experiments is difficult, computational methods could play a complementary role in elucidating the tertiary structure. Recently, we developed a new computational modeling method, IDP-LZerD, that can model complex structures with IDPs that are substantially longer than the usual lengths of peptides. Here, we discuss the ideas behind the IDP-LZerD pipeline with several examples.
Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) are fascinating dynamic conformational ensembles that are observed under physiological conditions. They facilitate a wide variety of biological processes via mechanisms that are distinct from their structured counterparts. Intrinsic disorder enables complex regulation using concerted molecular recognition, posttranslational modification, and alternative splicing. A broad analysis of IDRs reveals their central role in molecular recognition and cellular regulation. The diverse functions of IDRs are complemented by their diverse biophysical properties. Under the single moniker of disorder exist a variety of protein states that vary from protein-to-protein or for the same protein in different biological contexts. The biophysical and functional properties of IDRs are reflected in their composition, sequence complexity, and conservation. These sequence properties have been leveraged to design algorithms that accurately predict intrinsic disorder and certain molecular functions of disorder directly from the protein sequence.
This article continues a series of short comments on the paradoxes and wonders of the protein intrinsic disorder phenomenon by introducing the "stability of instability" paradox. Intrinsically disordered proteins (IDPs) are characterized by the lack of stable 3D-structure, and, as a result, have an exceptional ability to sustain exposure to extremely harsh environmental conditions (an illustration of the "you cannot break what is already broken" principle). Extended IDPs are known to possess extreme thermal and acid stability and are able either to keep their functionality under these extreme conditions or to rapidly regain their functionality after returning to the normal conditions. Furthermore, sturdiness of intrinsic disorder and its capability to "ignore" harsh conditions provides some interesting and important advantages to its carriers, at the molecular (e.g., the cell wall-anchored accumulation-associated protein playing a crucial role in intercellular adhesion within the biofilm of ), supramolecular (e.g., protein complexes, biologic liquid-liquid phase transitions, and proteinaceous membrane-less organelles), and organismal levels (e.g., the recently popularized case of the microscopic animals, tardigrades, or water bears, that use intrinsically disordered proteins to survive desiccation).
This is the 6th issue of the Digested Disorder series that continues to use only 2 criteria for inclusion of a paper to this digest: The publication date (a paper should be published within the covered time frame) and the topic (a paper should be dedicated to any aspect of protein intrinsic disorder). The current digest issue covers papers published during the second quarter of 2014; i.e., during the period of April, May, and June of 2014. Similar to previous issues, the papers are grouped hierarchically by topics they cover, and for each of the included papers a short description is given on its major findings.
This study was conducted to identify the source of animal meat based on the peculiarities of protein intrinsic disorder distribution in mitochondrial cytochrome b (mtCyt-b). The analysis revealed that animal and avian species can be discriminated based on the proportions of the two groups of residues, Leu+Ile, and Ser+Pro+Ala, in the amino acid sequences of their mtCyt-b. Although levels of the overall intrinsic disorder in mtCyt-b is not very high, the peculiarities of disorder distribution within the sequences of mtCyt-b from different species varies in a rather specific way. In fact, positions and intensities of disorder/flexibility "signals" in the corresponding disorder profiles are relatively unique for avian and animal species. Therefore, it is possible to devise a set of simple rules based on the peculiarities of disorder profiles of their mtCyt-b proteins to discriminate among species. This intrinsic disorder-based analysis represents a new technique that could be used to provide a promising solution for identification of the source of meats.
The abilities to crystalize of a globular protein and to solve its crystal structure seem to represent triumph of the lock-and-key model of protein functionality, where the presence of unique 3D structure resembling aperiodic crystal is considered as a prerequisite for a given protein to possess specific biologic activity. The history of protein crystallography has its roots in first crystal structures of myoglobin, lysozyme, RNase A, chymotrypsin, cytochrome c, and carboxypeptidase A1 solved more than 50 y ago. This article briefly considers extensive structural information currently available for these proteins and shows that the bottoms of their folding funnels (i.e., the lowest parts of their potential energy landscapes) are not smoothed but rugged. In other words, these crystallization classics are characterized by significant conformational flexibility and are not rigid (immobile) crystal-like entities.
Amyloid-β42 (Aβ42) is an intrinsically disordered peptide intimately related to the pathogenesis of several neurodegenerative diseases. Molecular dynamics (MD) simulations are extensively utilized in the characterization of the structures and conformational dynamics of intrinsically disordered proteins (IDPs) including Aβ42, with AMBER and CHARMM parameters being commonly used in these studies. Recently, comparison of the effects of force field parameters on the Aβ42 structures has started to gain significant attention. In this study, the structures of Aβ42 are simulated using AMBER FF99SB and CHARMM22/CMAP parameters via replica exchange MD simulations utilizing a widely used clustering algorithm. These analyses show that the structural properties (extent and positioning of the elements of secondary and tertiary structure), radius of gyration values, number and position of salt bridges are extremely dependent on the chosen force field parameters notably with the usage of clustering algorithms. For example, predicted secondary structure elements, which are of the great importance for better understanding of the molecular mechanisms of neurodegenerative diseases, deviate enormously in models generated using currently available force field parameters for proteins. Based on the derived models, chemical shift values are calculated and compared to the experimentally determined data. This comparison revealed that although both force field parameters yield results in agreement with experiments, the obtained structural properties were rather different using a clustering algorithm. In other words, these results show that the predicted structures depend heavily on the force field parameters. Importantly, since none of the force field parameters currently utilized in MD studies were developed specifically taking into account the disordered nature of IDPs, these findings clearly indicate that new force field parameters have to be developed for IDPs considering their rapid flexibility and dynamics with high amplitude. Furthermore, molecular simulations of IDPs are typically conducted using one water volume. We show that the confined aqueous volume impacts the predicted structural properties of Aβ42 in water. Although up to date, confined aqueous volume effects have been ignored in the MD simulations of IDPs in water, our data indicate that these effects have to be taken into account in predicting the structural and thermodynamic properties of disordered proteins in solution.
At first glance it may seem that intrinsically disordered proteins (IDPs) and IDP regions (IDPRs) are simpler than ordered proteins and domains on multiple levels. However, such multilevel simplicity equips these proteins with the ability to have very complex behavior.
We analyze a correlation between the GC content in genes of 12 eukaryotic species and the level of intrinsic disorder in their corresponding proteins. Comprehensive computational analysis has revealed that the disordered regions in eukaryotes are encoded by the GC-enriched gene regions and that this enrichment is correlated with the amount of disorder and is present across proteins and species characterized by varying amounts of disorder. The GC enrichment is a result of higher rate of amino acid coded by GC-rich codons in the disordered regions. Individual amino acids have the same GC-content profile between different species. Eukaryotic proteins with the disordered regions encoded by the GC-enriched gene segments carry out important biological functions including interactions with RNAs, DNAs, nucleotides, binding of calcium and metal ions, are involved in transcription, transport, cell division and certain signaling pathways, and are localized primarily in nucleus, cytosol and cytoplasm. We also investigate a possible relationship between GC content, intrinsic disorder and protein evolution. Analysis of a devised "age" of amino acids, their disorder-promoting capacity and the GC-enrichment of their codons suggests that the early amino acids are mostly disorder-promoting and their codons are GC-rich while most of late amino acids are mostly order-promoting.
This is the 5th issue of the Digested Disorder series that represents a reader's digest of the scientific literature on intrinsically disordered proteins. We continue to use only 2 criteria for inclusion of a paper to this digest: The publication date (a paper should be published within the covered time frame) and the topic (a paper should be dedicated to any aspect of protein intrinsic disorder). The current digest issue covers papers published during the first quarter of 2014; i.e., during the period of January, February, and March of 2014. Similar to previous issues, the papers are grouped hierarchically by topics they cover, and for each of the included papers a short description is given on its major findings.
