To be disordered or not to be disordered: is that still a question for proteins in the cell?

There is ample evidence that many proteins or regions of proteins lack a well-defined folded structure under native-like conditions. These are called intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs). Whether this intrinsic disorder is also their main structural characteristic in living cells has been a matter of intense debate. The structural analysis of IDPs became an important challenge also because of their involvement in a plethora of human diseases, which made IDPs attractive targets for therapeutic development. Therefore, biophysical approaches are increasingly being employed to probe the structural and dynamical state of proteins, not only in isolation in a test tube, but also in a complex biological environment and even within intact cells. Here, we survey direct and indirect evidence that structural disorder is in fact the physiological state of many proteins in the proteome. The paradigmatic case of α-synuclein is used to illustrate the controversial nature of this topic.     

Structural disorder in plant proteins: where plasticity meets sessility

Plants are sessile organisms. This intriguing nature provokes the question of how they survive despite the continual perturbations caused by their constantly changing environment. The large amount of knowledge accumulated to date demonstrates the fascinating dynamic and plastic mechanisms, which underpin the diverse strategies selected in plants in response to the fluctuating environment. This phenotypic plasticity requires an efficient integration of external cues to their growth and developmental programs that can only be achieved through the dynamic and interactive coordination of various signaling networks. Given the versatility of intrinsic structural disorder within proteins, this feature appears as one of the leading characters of such complex functional circuits, critical for plant adaptation and survival in their wild habitats. In this review, we present information of those intrinsically disordered proteins (IDPs) from plants for which their high level of predicted structural disorder has been correlated with a particular function, or where there is experimental evidence linking this structural feature with its protein function. Using examples of plant IDPs involved in the control of cell cycle, metabolism, hormonal signaling and regulation of gene expression, development and responses to stress, we demonstrate the critical importance of IDPs throughout the life of the plant.     

Evolution of intrinsic disorder in eukaryotic proteins

Conformational flexibility conferred though regions of intrinsic structural disorder allows proteins to behave as dynamic molecules. While it is well-known that intrinsically disordered regions can undergo disorder-to-order transitions in real-time as part of their function, we also are beginning to learn more about the dynamics of disorder-to-order transitions along evolutionary time-scales. Intrinsically disordered regions endow proteins with functional promiscuity, which is further enhanced by the ability of some of these regions to undergo real-time disorder-to-order transitions. Disorder content affects gene retention after whole genome duplication, but it is not necessarily conserved. Altered patterns of disorder resulting from evolutionary disorder-to-order transitions indicate that disorder evolves to modify function through refining stability, regulation, and interactions. Here, we review the evolution of intrinsically disordered regions in eukaryotic proteins. We discuss the interplay between secondary structure and disorder on evolutionary time-scales, the importance of disorder for eukaryotic proteome expansion and functional divergence, and the evolutionary dynamics of disorder.     

Scaffolding proteins and their role in viral assembly

Scaffolding proteins are proteins that are required to catalyse, regulate or modulate some step in the assembly of a macromolecular complex. They associate specifically with the nascent protein complex during assembly, but are subsequently removed, and are absent from the mature structure. Scaffolding proteins have been described primarily from viral systems, in particular from the double-stranded DNA bacteriophages, but most likely play a more general role in macromolecular assembly, a fundamental process in all biological systems. Scaffolding proteins may act in a specific fashion, by actively encouraging the formation of correct protein-protein interactions, or more generally by nucleating and promoting assembly. They may also work to ensure the fidelity of the assembly process by preventing the formation of improper interactions, in many ways similar to the role of molecular chaperones in protein folding. In viruses, scaffolding proteins are found both in the form of internal cores and external bracing, and may form elaborate and complex structures. This review will focus on the viral scaffolding proteins, for which an increasing amount of structural and functional information has recently become available.     

A creature with a hundred waggly tails: intrinsically disordered proteins in the ribosome

Intrinsic disorder (i.e., lack of a unique 3-D structure) is a common phenomenon, and many biologically active proteins are disordered as a whole, or contain long disordered regions. These intrinsically disordered proteins/regions constitute a significant part of all proteomes, and their functional repertoire is complementary to functions of ordered proteins. In fact, intrinsic disorder represents an important driving force for many specific functions. An illustrative example of such disorder-centric functional class is RNA-binding proteins. In this study, we present the results of comprehensive bioinformatics analyses of the abundance and roles of intrinsic disorder in 3,411 ribosomal proteins from 32 species. We show that many ribosomal proteins are intrinsically disordered or hybrid proteins that contain ordered and disordered domains. Predicted globular domains of many ribosomal proteins contain noticeable regions of intrinsic disorder. We also show that disorder in ribosomal proteins has different characteristics compared to other proteins that interact with RNA and DNA including overall abundance, evolutionary conservation, and involvement in protein–protein interactions. Furthermore, intrinsic disorder is not only abundant in the ribosomal proteins, but we demonstrate that it is absolutely necessary for their various functions.     

Protein intrinsic disorder as a flexible armor and a weapon of HIV-1

Many proteins and protein regions are disordered in their native, biologically active states. These proteins/regions are abundant in different organisms and carry out important biological functions that complement the functional repertoire of ordered proteins. Viruses, with their highly compact genomes, small proteomes, and high adaptability for fast change in their biological and physical environment utilize many of the advantages of intrinsic disorder. In fact, viral proteins are generally rich in intrinsic disorder, and intrinsically disordered regions are commonly used by viruses to invade the host organisms, to hijack various host systems, and to help viruses in accommodation to their hostile habitats and to manage their economic usage of genetic material. In this review, we focus on the structural peculiarities of HIV-1 proteins, on the abundance of intrinsic disorder in viral proteins, and on the role of intrinsic disorder in their functions.     

How order and disorder within paramyxoviral nucleoproteins and phosphoproteins orchestrate the molecular interplay of transcription and replication

In this review, we summarize computational and experimental data gathered so far showing that structural disorder is abundant within paramyxoviral nucleoproteins (N) and phosphoproteins (P). In particular, we focus on measles, Nipah, and Hendra viruses and highlight both commonalities and differences with respect to the closely related Sendai virus. The molecular mechanisms that control the disorder-to-order transition undergone by the intrinsically disordered C-terminal domain (N_TAIL) of their N proteins upon binding to the C-terminal X domain (XD) of the homologous P proteins are described in detail. By having a significant residual disorder, N_TAIL–XD complexes are illustrative examples of “fuzziness”, whose possible functional significance is discussed. Finally, the relevance of N–P interactions as promising targets for innovative antiviral approaches is underscored, and the functional advantages of structural disorder for paramyxoviruses are pinpointed.     

Behaviour of intrinsically disordered proteins in protein–protein complexes with an emphasis on fuzziness

Intrinsically disordered proteins (IDPs) do not, by themselves, fold into a compact globular structure. They are extremely dynamic and flexible, and are typically involved in signalling and transduction of information through binding to other macromolecules. The reason for their existence may lie in their malleability, which enables them to bind several different partners with high specificity. In addition, their interactions with other macromolecules can be regulated by a variable amount of chemically diverse post-translational modifications. Four kinetically and energetically different types of complexes between an IDP and another macromolecule are reviewed: (1) simple two-state binding involving a single binding site, (2) avidity, (3) allovalency and (4) fuzzy binding; the last three involving more than one site. Finally, a qualitative definition of fuzzy binding is suggested, examples are provided, and its distinction to allovalency and avidity is highlighted and discussed.     

Conditionally disordered proteins: bringing the environment back into the fold

For many proteins, biological function requires the folding of the polypeptide chain into a unique and persistent tertiary structure. This review concerns proteins that adopt a specific tertiary structure to function, but are otherwise partially or completely disordered. The biological cue for protein folding is environmental perturbation or minor post-translational modification. Hence, we term these proteins conditionally disordered. Many of these proteins recognize and bind other molecules, and conditional disorder has been hypothesized to allow for more nuanced control and regulation of binding processes. However, this remains largely unproven. The sequences of conditionally disordered proteins suggest their propensity to fold; yet, under the standard laboratory conditions, they do not do so, which may appear surprising. We argue that the surprise results from the failure to consider the role of the environment in protein structure formation and that conditional disorder arises as a natural consequence of the marginal stability of the folded state.     

Intrinsic disorder here,there, and everywhere,and nowhere to escape from it

The concept of protein intrinsic disorder persistently penetrates into all areas of modern protein science. It cannot be ignored anymore, and cannot be shrugged off, as it represents a vital feature (or, more correctly, a broad spectrum of important features), which, when added to and mixed with features arising from the well established protein structure-function paradigm, complete the picture of a functioning protein. The field of protein intrinsic disorder is very dynamic and fast developing. This Multi-Author Review represents a snapshot of this field by introducing some recent advances. Articles assembled in this Multi-Author Review introduce some of the new aspects of intrinsic disorder, outline some fascinating ideas related to the intrinsically disordered proteins, their structure, and functionality, and show challenges related to the analysis of proteins carrying intrinsic disorder.     

A conformationally isoformic thermophilic protein with high kinetic unfolding barriers

The basis for the stability of thermophilic proteins is of fundamental interest for extremophile biology. We investigated the folding and unfolding processes of the homotetrameric Thermoanaerobacter brockii alcohol dehydrogenase (TBADH). TBADH subunits were 4.8 kcal/mol less stable towards guanidinium chloride (GdmCl) unfolding compared to urea, indicating ionic modulation of TBADH stability. Strongly denaturing conditions promoted mono-exponential unfolding kinetics with linear dependence on denaturant concentration. Here TBADH unfolded >40-fold slower when extrapolated from urea as compared to GdmCl unfolding. A marked unfolding hysteresis was shown when comparing refolding and unfolding in urea. An unusual biphasic unfolding trajectory with an exceptionally slow phase at intermediate concentrations of GdmCl and urea was also observed. We advocate that TBADH forms two distinctly different tetrameric isoforms, and likely an ensemble of native states. This unusual supramolecular folding behavior has been shown responsible for formation of amyloidotic yeast prion strains and can have functional importance for TBADH. Received 9 November 2007; received after revision 19 December 2007; accepted 8 January 2008     

Molecular guardians for newborn proteins: ribosome-associated chaperones and their role in protein folding

A central dogma in biology is the conversion of genetic information into active proteins. The biosynthesis of proteins by ribosomes and the subsequent folding of newly made proteins represent the last crucial steps in this process. To guarantee the correct folding of newly made proteins, a complex chaperone network is required in all cells. In concert with ongoing protein biosynthesis, ribosome-associated factors can interact directly with emerging nascent polypeptides to protect them from degradation or aggregation, to promote folding into their native structure, or to otherwise contribute to their folding program. Eukaryotic cells possess two major ribosome-associated systems, an Hsp70/Hsp40-based chaperone system and the functionally enigmatic NAC complex, whereas prokaryotes employ the Trigger Factor chaperone. Recent structural insights into Trigger Factor reveal an intricate cradle-like structure that, together with the exit site of the ribosome, forms a protected environment for the folding of newly synthesized proteins. Received 29 June 2005; received after revision 4 August 2005; accepted 18 August 2005     

Biochemical,biophysical and molecular dynamics studies on the proteoglycan-like domain of carbonic anhydrase IX

Human carbonic anhydrase IX (hCA IX) is a tumour-associated enzyme present in a limited number of normal tissues, but overexpressed in several malignant human tumours. It is a transmembrane protein, where the extracellular region consists of a greatly investigated catalytic CA domain and a much less investigated proteoglycan-like (PG) domain. Considering its important role in tumour biology, here, we report for the first time the full characterization of the PG domain, providing insights into its structural and functional features. In particular, this domain has been produced at high yields in bacterial cells and characterized by means of biochemical, biophysical and molecular dynamics studies. Results show that it belongs to the family of intrinsically disordered proteins, being globally unfolded with only some local residual polyproline II secondary structure. The observed conformational flexibility may have several important roles in tumour progression, facilitating interactions of hCA IX with partner proteins assisting tumour spreading and progression.     

Structure and function of the GroE chaperone

The Escherichia coli proteins GroEL and GroES were the first chaperones to be studied in detail and have thus become a role model for assisted protein folding in general. A wealth of both structural and functional data on the GroE system has been accumulated over the past years, enabling us now to understand the basic principles of how this fascinating protein-folding machine accomplishes its task. According to the current model, GroE processes a nonnative polypeptide in a cycle consisting of three steps. First, the polypeptide substrate is captured by GroEL. Upon binding of the co-chaperone GroES and ATP, the substrate is then discharged into a unique microenvironment inside of the chaperone, which promotes productive folding. After hydrolysis of ATP, the polypeptide is released into solution. Moreover, GroE may actively increase the folding efficiency, e.g. by unfolding of misfolded protein molecules. The mechanisms underlying these features, however, are yet not well characterized.     

Functions of intrinsic disorder in transmembrane proteins

Intrinsic disorder is common in integral membrane proteins, particularly in the intracellular domains. Despite this observation, these domains are not always recognized as being disordered. In this review, we will discuss the biological functions of intrinsically disordered regions of membrane proteins, and address why the flexibility afforded by disorder is mechanistically important. Intrinsically disordered regions are present in many common classes of membrane proteins including ion channels and transporters; G-protein coupled receptors (GPCRs), receptor tyrosine kinases and cytokine receptors. The functions of the disordered regions are many and varied. We will discuss selected examples including: (1) Organization of receptors, kinases, phosphatases and second messenger sources into signaling complexes. (2) Modulation of the membrane-embedded domain function by ball-and-chain like mechanisms. (3) Trafficking of membrane proteins. (4) Transient membrane associations. (5) Post-translational modifications most notably phosphorylation and (6) disorder-linked isoform dependent function. We finish the review by discussing the future challenges facing the membrane protein community regarding protein disorder.     

Protein folding in membranes

Separation of cells and organelles by bilayer membranes is a fundamental principle of life. Cellular membranes contain a baffling variety of proteins, which fulfil vital functions as receptors and signal transducers, channels and transporters, motors and anchors. The vast majority of membrane-bound proteins contain bundles of α-helical transmembrane domains. Understanding how these proteins adopt their native, biologically active structures in the complex milieu of a membrane is therefore a major challenge in today’s life sciences. Here, we review recent progress in the folding, unfolding and refolding of α-helical membrane proteins and compare the molecular interactions that stabilise proteins in lipid bilayers. We also provide a critical discussion of a detergent denaturation assay that is increasingly used to determine membrane-protein stability but is not devoid of conceptual difficulties.     

Amyloid fibrils from the viewpoint of protein folding

In amyloid related diseases, proteins form fibrillar aggregates with highly ordered -sheet structure regardless of their native conformations. Formation of such amyloid fibrils can be reproducible in vitro using isolated proteins/peptides, suggesting that amyloid fibril formation takes place as a result of protein conformational change. In vitro studies revealed that perturbation of the native structure is important for the fibril formation, and it is suggested that the mechanisms of amyloid fibril formation share the mechanisms of protein folding. In particular, amyloid fibril formation is similar to one of the common features of proteins, i.e. amorphous aggregation upon partial unfolding, which is likely driven by hydrophobic interactions through exposed protein interior. However, these molecular associations are distinct phenomena, and identifying factors that lead to amyloid fibril formation would precede our understanding of the mechanisms of amyloid fibrillization. The necessity of understanding the nature of protein denatured states is also suggested.Received 6 July 2003; accepted 19 August 2003     

Methods of probing the interactions between small molecules and disordered proteins

It is generally recognized that a large fraction of the human proteome is made up of proteins that remain disordered in their native states. Despite the fact that such proteins play key biological roles and are involved in many major human diseases, they still represent challenging targets for drug discovery. A major bottleneck for the identification of compounds capable of interacting with these proteins and modulating their disease-promoting behaviour is the development of effective techniques to probe such interactions. The difficulties in carrying out binding measurements have resulted in a poor understanding of the mechanisms underlying these interactions. In order to facilitate further methodological advances, here we review the most commonly used techniques to probe three types of interactions involving small molecules: (1) those that disrupt functional interactions between disordered proteins; (2) those that inhibit the aberrant aggregation of disordered proteins, and (3) those that lead to binding disordered proteins in their monomeric states. In discussing these techniques, we also point out directions for future developments.