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.     

Order propensity of an intrinsically disordered protein,the cyclin‐dependent‐kinase inhibitor Sic1

Intrinsically disordered proteins (IDPs) carry out important biological functions and offer an instructive model system for folding and binding studies. However, their structural characterization in the absence of interactors is hindered by their highly dynamic conformation. The cyclin‐dependent‐kinase inhibitor (Cki) Sic1 from Saccharomyces cerevisiae is a key regulator of the yeast cell cycle, which controls entrance into S phase and coordination between cell growth and proliferation. Its last 70 out of 284 residues display functional and structural homology to the inhibitory domain of mammalian p21 and p27. Sic1 has escaped systematic structural characterization until now. Here, complementary biophysical methods are applied to the study of conformational properties of pure Sic1 in solution. Based on sequence analysis, gel filtration, circular dichroism (CD), electrospray‐ionization mass spectrometry (ESI‐MS), and limited proteolysis, it can be concluded that the whole molecule exists in a highly disordered state and can, therefore, be classified as an IDP. However, the results of these experiments indicate, at the same time, that the protein displays some content in secondary and tertiary structure, having properties similar to those of molten globules or premolten globules. Proteolysis‐hypersensitive sites cluster at the N‐terminus and in the middle of the molecule, whereas the most structured region resides at the C‐terminus, including part of the inhibitory domain and the casein‐kinase‐2 (CK2) phosphorylation target S201. The mutations S201A and S201E, which are known to affect Sic1 function, do not have significant effects on the conformational properties of the pure protein. Proteins 2009;76:731–746. © 2009 Wiley‐Liss, Inc.     

Denaturant mediated unfolding of both native and molten globule states of maltose binding protein are accompanied by large deltaCp's.

Maltose binding protein (MBP) is a large, monomeric two domain protein containing 370 amino acids. In the absence of denaturant at neutral pH, the protein is in the native state, while at pH 3.0 it forms a molten globule. The molten globule lacks a tertiary circular dichroism signal but has secondary structure similar to that of the native state. The molten globule binds 8-anilino-1-naphthalene sulfonate (ANS). The unfolding thermodynamics of MBP at both pHs were measured by carrying out a series of isothermal urea melts at temperatures ranging from 274-329 K. At 298 K, values of deltaGdegrees , deltaCp, and Cm were 3.1+/-0.2 kcal mol(-1), 5.9+/-0.8 kcal mol(-1) K(-1) (15.9 cal (mol-residue)(-1) K(-1)), and 0.8 M, respectively, at pH 3.0 and 14.5+/-0.4 kcal mol(-1), 8.3+/-0.7 kcal mol(-1) K(-1) (22.4 kcal (mol-residue)(-1) K(-1)), and 3.3 M, respectively, at pH 7.1. Guanidine hydrochloride denaturation at pH 7.1 gave values of deltaGdegrees and deltaCp similar to those obtained with urea. The m values for denaturation are strongly temperature dependent, in contrast to what has been previously observed for small globular proteins. The value of deltaCp per mol-residue for the molten globule is comparable to corresponding values of deltaCp for the unfolding of typical globular proteins and suggests that it is a highly ordered structure, unlike molten globules of many small proteins. The value of deltaCp per mol-residue for the unfolding of the native state is among the highest currently known for any protein.     

Operational definition of intrinsically unstructured protein sequences based on susceptibility to the 20S proteasome

Intrinsically unstructured proteins (IUPs), also known as natively unfolded proteins, lack well-defined secondary and tertiary structure under physiological conditions. In recent years, growing experimental and theoretical evidence has accumulated, indicating that many entire proteins and protein sequences are unstructured under physiological conditions, and that they play significant roles in diverse cellular processes. Bioinformatic algorithms have been developed to identify such sequences in proteins for which structural data are lacking, but still generate substantial numbers of false positives and negatives. We describe here a simple and reliable in vitro assay for identifying IUP sequences based on their susceptibility to 20S proteasomal degradation. We show that 20S proteasomes digest IUP sequences, under conditions in which native, and even molten globule states, are resistant. Furthermore, we show that protein-protein interactions can protect IUPs against 20S proteasomal action. Taken together, our results thus suggest that the 20S proteasome degradation assay provides a powerful system for operational definition of IUPs.     

Huntingtin interacting protein HYPK is intrinsically unstructured

To characterize HYPK, originally identified as a novel huntingtin (Htt) interacting partner by yeast two hybrid assay, we used various biophysical and biochemical techniques. The molecular weight of the protein, determined by gel electrophoresis, was found to be about 1.3-folds ( approximately 22 kDa) higher than that obtained from mass spectrometric analysis (16.9 kDa). In size exclusion chromatography experiment, HYPK was eluted in three fractions, the hydrodynamic radii for which were calculated to be approximately 1.5-folds (23.06 A) higher than that expected for globular proteins of equivalent mass (17.3 A). The protein exhibited predominantly (63%) random coil characteristics in circular dichroism spectroscopy and was highly sensitive to limited proteolysis by trypsin and papain, indicating absence of any specific domain. Experimental evidences with theoretical analyses of amino acids composition of HYPK and comparison with available published data predicts that HYPK is an intrinsically unstructured protein (IUP) with premolten globule like conformation. In presence of increasing concentration of Ca(2+), HYPK showed conformational alterations as well as concomitant reduction of hydrodynamic radius. Even though any link between the natively unfolded nature of HYPK, its conformational sensitivity towards Ca(2+) and interaction with Htt is yet to be established, its possible involvement in Huntington's disease pathogenesis is discussed.     

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.     

Denatured collapsed states in protein folding: example of apomyoglobin.

Experimental approaches, including circular dichroism, small angle X-ray scattering, steady-state fluorescence, and fluorescence energy transfer, were applied to study the 3D-structure of apomyolgobin in different conformational states. These included the native and molten globules, along with either less ordered conformations induced by the addition of anions or completely unfolded states. The results show that the partially folded forms of apomyoglobin stabilized by KCl and/or Na(2)SO(4) under unfolding conditions (pH 2) exhibit a significant amount of secondary structure (circular dichroism), low packing density of protein molecules (SAXS), and native-like dimensions of the AGH core (fluorescence energy transfer). This finding indicates that a native-like tertiary fold of the polypeptide chain, i.e., the spatial organization of secondary structure elements, most likely emerges prior to the formation of the molten globule state.     

Natively unfolded C-terminal domain of caldesmon remains substantially unstructured after the effective binding to calmodulin

The structure of C-terminal domain (CaD136, C-terminal residues 636-771) of chicken gizzard caldesmon has been analyzed by a variety of physico-chemical methods. We are showing here that CaD136 does not have globular structure, has low secondary structure content, is essentially noncompact, as it follows from high R(g) and R(S) values, and is characterized by the absence of distinct heat absorption peaks, i.e. it belongs to the family of natively unfolded (or intrinsically unstructured) proteins. Surprisingly, effective binding of single calmodulin molecule (K(d) = 1.4 +/- 0.2 microM) leads only to a very moderate folding of this protein and CaD136 remains substantially unfolded within its tight complex with calmodulin. The biological significance of these observations is discussed.     

Role of quaternary structure in muscle creatine kinase stability: Tryptophan 210 is important for dimer cohesion

A mutant of the dimeric rabbit muscle creatine kinase (MM-CK) in which tryptophan 210 was replaced has been studied to assess the role of this residue in dimer cohesion and the importance of the dimeric state for the native enzyme stability. Wild-type protein equilibrium unfolding induced by guanidine hydrochloride occurs through intermediate states with formation of a molten globule and a premolten globule. Unlike the wild-type enzyme, the mutant inactivates at lower denaturant concentration and the loss of enzymatic activity is accompanied by the dissociation of the dimer into two apparently compact monomers. However, the Stokes radius of the monomer increases with denaturant concentration as determined by size exclusion chromatography, indicating that, upon monomerization, the protein structure is destabilized. Binding of 8-anilinonaphthalene-1-sulfonate shows that the dissociated monomer exposes hydrophobic patches at its surface, suggesting that it could be a molten globule. At higher denaturant concentrations, both wild-type and mutant follow similar denaturation pathways with formation of a premolten globule around 1.5-M guanidine, indicating that tryptophan 210 does not contribute to a large extent to the monomer conformational stability, which may be ensured in the dimeric state through quaternary interactions. Proteins 32:43–51, 1998. © 1998 Wiley-Liss, Inc.     

Preformed structural elements feature in partner recognition by intrinsically unstructured proteins

Intrinsically unstructured proteins (IUPs) are devoid of extensive structural order but often display signs of local and limited residual structure. To explain their effective functioning, we reasoned that such residual structure can be crucial in their interactions with their structured partner(s) in a way that preformed structural elements presage their final conformational state. To check this assumption, a database of 24 IUPs with known 3D structures in the bound state has been assembled and the distribution of secondary structure elements and backbone torsion angles have been analysed. The high proportion of residues in coil conformation and with phi, psi angles in the disallowed regions of the Ramachandran map compared to the reference set of globular proteins shows that IUPs are not fully ordered even in their bound form. To probe the effect of partner proteins on IUP folding, inherent conformational preferences of IUP sequences have been assessed by secondary structure predictions using the GOR, ALB and PROF algorithms. The accuracy of predicting secondary structure elements of IUPs is similar to that of their partner proteins and is significantly higher than the corresponding values for random sequences. We propose that strong conformational preferences mark regions in IUPs (mostly helices), which correspond to their final structural state, while regions with weak conformational preferences represent flexible linkers between them. In our interpretation, preformed elements could serve as initial contact points, the binding of which facilitates the reeling of the flexible regions onto the template. This finding implies that IUPs draw a functional advantage from preformed structural elements, as they enable their facile, kinetically and energetically less demanding, interaction with their physiological partner.     

Gir2 is an intrinsically unstructured protein that is present in Saccharomyces cerevisiae as a group of heterogeneously electrophoretic migrating forms

Gir2 is a highly acidic cytoplasmic protein of Saccharomyces cerevisiae of unknown function that shows an anomalous migration on SDS-PAGE. Based on its large Stokes radius and thermostability, we have previously suggested that Gir2 lacks extensive secondary structure. Here we report that Gir2 is extremely sensitive to proteolysis when compared to glutathione-S-transferase, a highly structured protein, further indicating its unfolded nature. Prediction based on the FoldIndex program also indicates that Gir2 is a disordered protein. Using truncated forms of Gir2 we show that the N-terminal half of this protein, with its high content of acidic amino acid residues, is responsible for the anomalous electrophoretic behavior of Gir2. Because all these features are hallmarks of intrinsically unstructured proteins (IUP), we propose that Gir2 is another representative of the IUP group of proteins. Additionally, we describe that the endogenous yeast Gir2 shows heterogeneous electrophoretic mobility, which is not due to proteolytic cleavage.     

A novel approach to segregate and identify functional loop regions in protein structures using their Ramachandran maps

The loops which connect or flank helices/sheets in protein structures are known to be functionally important. However, ironically they also belong to the part of protein whose structure is least accurately predicted. Here, a new method to isolate and analyze loop regions in protein structure is proposed using the spatial coordinates of the solved three‐dimensional structure. The extent of dispersion among points of successive amino acid residues in the Ramachandran map of protein region is utilized to calculate the Mean Separation between these points in the Ramachandran Plot (MSRP). Based on analysis of 2935 protein secondary structure regions obtained using DSSP software, spanning a range from 2 to 64 residues, taken from a set of 170 proteins, it is shown that helices (MSRP < 17) and strands (MSRP < 64) stand effectively demarcated from the loop regions (MSRP > 130). Analysis of 43 DNA binding and 98 ligand binding proteins revealed several loop regions with clear change in MSRP subsequent to binding. The population of such loops correlated with the magnitude of backbone displacement in the protein subsequent to binding. Can changes in MSRP quantify the temporal oscillations in dihedral angles among structured/unstructured regions in proteins? Molecular dynamics simulations (10 ns) revealed that deviations in MSRP among different snapshots in the trajectory were at least twofold higher for unstructured proteins in comparison with ordered proteins. The above results validate the use of MSRP parameter as a tool to identify and investigate functionally active loops and unstructured regions in protein structures. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Structural energetics of the molten globule state

Certain partly ordered protein conformations, commonly called “moltenglobule states,” are widely believed to represent protein folding intermediates. Recentstructural studies of molten globule states ofdifferent proteins have revealed features whichappear to be general in scope. The emergingconsensus is that these partly ordered forms exhibit a high content of secondary structure, considerable compactness, nonspecific tertiary structure, and significant structural flexibility. These characteristics may be used to define ageneral state of protein folding called “the molten globule state,” which is structurally andthermodynamically distinct from both the native state and the denatured state. Despite exaatensive knowledge of structural features of afew molten globule states, a cogent thermodynamic argument for their stability has not yetbeen advanced. The prevailing opinion of thelast decade was that there is little or no enthalpy difference or heat capacity differencebetween the molten globule state and the unfolded state. This view, however, appears to beat variance with the existing database of protein structural energetics and with recent estimates of the energetics of denaturation of α-lactalbumin, cytochrome c, apomyoglobin, and T4 lysozyme. We discuss these four proteins at length. The results of structural studies, together with the existing thermodynamic values for fundamental interactions in proteins, provide the foundation for a structural thermodynamic framework which can account for the observed behavior of molten globule states. Within this framework, we analyze the physical basis for both the high stability of several molten globule states and the low probability of other protential folding intermediates. Additionally, we consider, in terms of reduced enthalpy changes and disrupted cooperative interactions, the thermodynamic basis for the apparent absence of a thermally induced, cooperative unfolding transition for some molten globule states. © 1993 Wiley-Liss, Inc.     

Intrinsically Semi-disordered State and Its Role in Induced Folding and Protein Aggregation

Intrinsically disordered proteins (IDPs) refer to those proteins without fixed three-dimensional structures under physiological conditions. Although experiments suggest that the conformations of IDPs can vary from random coils, semi-compact globules, to compact globules with different contents of secondary structures, computational efforts to separate IDPs into different states are not yet successful. Recently, we developed a neural-network-based disorder prediction technique SPINE-D that was ranked as one of the top performing techniques for disorder prediction in the biannual meeting of critical assessment of structure prediction techniques (CASP 9, 2010). Here, we further analyze the results from SPINE-D prediction by defining a semi-disordered state that has about 50 % predicted probability to be disordered or ordered. This semi-disordered state is partially collapsed with intermediate levels of predicted solvent accessibility and secondary structure content. The relative difference in compositions between semi-disordered and fully disordered regions is highly correlated with amyloid aggregation propensity (a correlation coefficient of 0.86 if excluding four charged residues and proline, 0.73 if not). In addition, we observed that some semi-disordered regions participate in induced folding, and others play key roles in protein aggregation. More specifically, a semi-disordered region is amyloidogenic in fully unstructured proteins (such as alpha-synuclein and Sup35) but prone to local unfolding that exposes the hydrophobic core to aggregation in structured globular proteins (such as SOD1 and lysozyme). A transition from full disorder to semi-disorder at about 30–40 Qs is observed in the poly-Q (poly-glutamine) tract of huntingtin. The accuracy of using semi-disorder to predict binding-induced folding and aggregation is compared with several methods trained for the purpose. These results indicate the usefulness of three-state classification (order, semi-disorder, and full-disorder) in distinguishing nonfolding from induced-folding and aggregation-resistant from aggregation-prone IDPs and in locating weakly stable, locally unfolding, and potentially aggregation regions in structured proteins. A comparison with five representative disorder-prediction methods showed that SPINE-D is the only method with a clear separation of semi-disorder from ordered and fully disordered states.     

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.     

Polymeric aspects of protein folding: a brief overview

Regardless of the differences in primary amino acid sequences, protein molecules in a number of conformational states behave as polymer homologues, allowing speculations as to the volume interactions being a driving force in formation of equilibrium structures. For instance, both native and molten globules exhibit key features of polymer globules, where the fluctuations of the molecular density are expected to be much less than the molecular density itself. Protein molecules in the compact denatured (pre-molten globule) states possess properties of squeezed coils. In fact, even high concentrations of strong denaturants (e.g., urea and GdmCl) more likely constitute bad solvents for protein chains. Thus, globular proteins are probably never random coils without positional correlations and biological polypeptide chains represent the macromolecular coils below a critical point even under harsh denaturing conditions. Several implications of these findings to protein folding are discussed.     

DSC studies of a family of natively disordered fragments from Escherichia coli thioredoxin: surface burial in intrinsic coils

The accumulating data from proteome analysis indicates that numerous proteins have segments and/or domains, involved in regulatory functions of the eukaryotic cell, which are entirely unstructured under physiological conditions, challenging the structure-function paradigm. Although many such natively unfolded proteins have been structurally analyzed by NMR spectroscopy, little is known about solvent inaccessible surfaces in premolten globules and intrinsic coils. Recent DSC studies of two protein fragments have shown a promising way to estimate the predominantly hydrophobic buried surfaces [Georgescu, R. E., García-Mira, M. M., Tasayco, M. L., and Sánchez-Ruiz, J. M. (2001) Eur. J. Biochem. 268, 1-10]. Here we report a systematic heat capacity analysis of a family of natively disordered complementary fragments of oxidized Escherichia coli thioredoxin (1-31/32-108, 1-37/38-108, 1-50/51-108, and 38-73) which provides insights into the local and nonlocal interactions contributing to the burial of predominantly hydrophobic surface in intrinsic coils.     

Structural characterization of the molten globule of alpha-lactalbumin by solution X-ray scattering.

A compact denatured state is often observed under a mild denaturation condition for various proteins. A typical example is the alpha-lactalbumin molten globule. Although the molecular compactness and shape are the essential properties for defining the molten globule, there have been ambiguities of these properties for the molten globule of alpha-lactalbumin. Using solution X-ray scattering, we have examined the structural properties of two types of molten globule of alpha-lactalbumin, the apo-protein at neutral pH and the acid molten globule. The radius of gyration for the native holo-protein was 15.7 A, but the two different molten globules both had a radius of gyration of 17.2 A. The maximum dimension of the molecule was also increased from 50 A for the native state to 60 A for the molten globule. These values clearly indicate that the molten globule is not as compact as the native state. The increment in the radius of gyration was less than 10% for the alpha-lactalbumin molten globule, compared with up to 30% for the molten globules of other globular proteins. Intramolecular disulfide bonds restrict the molecular expansion of the molten globule. The distance distribution function of the alpha-lactalbumin molten globule is composed of a single peak suggesting a globular shape, which is simply swollen from the native state. The scattering profile in the high Q region of the molten globule indicates the presence of a significant amount of tertiary fold. Based on the structural properties obtained by solution X-ray scattering, general and conceptual structural images for the molten globules of various proteins are described and compared with the individual, detailed structural model obtained by nuclear magnetic resonance.     

Advantages of proteins being disordered

The past decade has witnessed great advances in our understanding of protein structure‐function relationships in terms of the ubiquitous existence of intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs). The structural disorder of IDPs/IDRs enables them to play essential functions that are complementary to those of ordered proteins. In addition, IDPs/IDRs are persistent in evolution. Therefore, they are expected to possess some advantages over ordered proteins. In this review, we summarize and survey nine possible advantages of IDPs/IDRs: economizing genome/protein resources, overcoming steric restrictions in binding, achieving high specificity with low affinity, increasing binding rate, facilitating posttranslational modifications, enabling flexible linkers, preventing aggregation, providing resistance to non‐native conditions, and allowing compatibility with more available sequences. Some potential advantages of IDPs/IDRs are not well understood and require both experimental and theoretical approaches to decipher. The connection with protein design is also briefly discussed.     

Non-Sequence-Specific Interactions Can Account for the Compaction of Proteins Unfolded under “Native” Conditions

Proteins unfolded by high concentrations of chemical denaturants adopt expanded, largely structure-free ensembles of conformations that are well approximated as random coils. In contrast, globular proteins unfolded under less denaturing conditions (via mutations, or transiently unfolded after a rapid jump to native conditions) and molten globules (arising due to mutations or cosolvents) are often compact. Here we explore the origins of this compaction using a truncated equilibrium-unfolded variant of the 57-residue FynSH3 domain. As monitored by far-UV circular dichroism, NMR spectroscopy, and hydrogen-exchange kinetics, CΔ4 (a 4-residue carboxy-terminal deletion variant of FynSH3) appears to be largely unfolded even in the absence of denaturant. Nevertheless, CΔ4 is quite compact under these conditions, with a hydrodynamic radius only slightly larger than that of the native protein. In order to understand the origins of this molten-globule-like compaction, we have characterized a random sequence polypeptide of identical amino acid composition to CΔ4. Notably, we find that the hydrodynamic radius of this random sequence polypeptide also approaches that of the native protein. Thus, while native-like interactions may contribute to the formation of compact “unfolded” states, it appears that non-sequence-specific monomer-monomer interactions can also account for the dramatic compaction observed for molten globules and the “physiological” unfolded state.