Long indels are disordered: A study of disorder and indels in homologous eukaryotic proteins

Proteins evolve through point mutations as well as by insertions and deletions (indels). During the last decade it has become apparent that protein regions that do not fold into three-dimensional structures, i.e. intrinsically disordered regions, are quite common. Here, we have studied the relationship between protein disorder and indels using HMM–HMM pairwise alignments in two sets of orthologous eukaryotic protein pairs. First, we show that disordered residues are much more frequent among indel residues than among aligned residues and, also are more prevalent among indels than in coils. Second, we observed that disordered residues are particularly common in longer indels. Disordered indels of short-to-medium size are prevalent in the non-terminal regions of proteins while the longest indels, ordered and disordered alike, occur toward the termini of the proteins where new structural units are comparatively well tolerated. Finally, while disordered regions often evolve faster than ordered regions and disorder is common in indels, there are some previously recognized protein families where the disordered region is more conserved than the ordered region. We find that these rare proteins are often involved in information processes, such as RNA processing and translation. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.     

Understanding protein non-folding

This review describes the family of intrinsically disordered proteins, members of which fail to form rigid 3-D structures under physiological conditions, either along their entire lengths or only in localized regions. Instead, these intriguing proteins/regions exist as dynamic ensembles within which atom positions and backbone Ramachandran angles exhibit extreme temporal fluctuations without specific equilibrium values. Many of these intrinsically disordered proteins are known to carry out important biological functions which, in fact, depend on the absence of a specific 3-D structure. The existence of such proteins does not fit the prevailing structure–function paradigm, which states that a unique 3-D structure is a prerequisite to function. Thus, the protein structure–function paradigm has to be expanded to include intrinsically disordered proteins and alternative relationships among protein sequence, structure, and function. This shift in the paradigm represents a major breakthrough for biochemistry, biophysics and molecular biology, as it opens new levels of understanding with regard to the complex life of proteins. This review will try to answer the following questions: how were intrinsically disordered proteins discovered? Why don't these proteins fold? What is so special about intrinsic disorder? What are the functional advantages of disordered proteins/regions? What is the functional repertoire of these proteins? What are the relationships between intrinsically disordered proteins and human diseases?     

Using Bayesian multinomial classifier to predict whether a given protein sequence is intrinsically disordered

Intrinsically disordered proteins (IDPs) lack a well-defined three-dimensional structure under physiological conditions. Intrinsic disorder is a common phenomenon, particularly in multicellular eukaryotes, and is responsible for important protein functions including regulation and signaling. Many disease-related proteins are likely to be intrinsically disordered or to have disordered regions. In this paper, a new predictor model based on the Bayesian classification methodology is introduced to predict for a given protein or protein region if it is intrinsically disordered or ordered using only its primary sequence. The method allows to incorporate length-dependent amino acid compositional differences of disordered regions by including separate statistical representations for short, middle and long disordered regions. The predictor was trained on the constructed data set of protein regions with known structural properties. In a Jack-knife test, the predictor achieved the sensitivity of 89.2% for disordered and 81.4% for ordered regions. Our method outperformed several reported predictors when evaluated on the previously published data set of Prilusky et al. [2005. FoldIndex: a simple tool to predict whether a given protein sequence is intrinsically unfolded. Bioinformatics 21 (16), 3435-3438]. Further strength of our approach is the ease of implementation.     

Limited proteolysis of natively unfolded protein 4E‐BP1 in the presence of trifluoroethanol

Natively unfolded (intrinsically disordered (ID) proteins) have been attracting an increasing attention due to their involvement in many regulatory processes. Natively unfolded proteins can fold upon binding to their metabolic partners. Coupled folding and binding events usually involve only relatively short motifs (binding motifs). These binding motifs which are able to fold should have an increased propensity to form a secondary structure. The aim of the present work was to probe the conformation of the intrinsically disordered protein 4E‐BP1 in the native and partly folded states by limited proteolysis and to reveal regions with a high propensity to form an ordered structure. Trifuoroethanol (TFE) in low concentrations (up to 15 vol%) was applied to increase the helical population of protein regions with a high intrinsic propensity to fold. When forming helical structures, these regions lose mobility and become more protected from proteases than random/unfolded protein regions. Limited proteolysis followed by mass spectrometry analysis allows identification of the regions with decreased mobility in TFE solutions. Trypsin and V8 proteases were used to perform limited proteolysis of the 4E‐BP1 protein in buffer and in solutions with low TFE concentrations at 37°C and at elevated temperatures (42 and 50°C). Comparison of the results obtained with the previously established 4E‐BP1 structure and the binding motif illustrates the ability of limited proteolysis in the presence of a folding assistant (TFE) to map the regions with high and low propensities to form a secondary structure revealing potential binding motifs inside the intrinsically disordered protein. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 591–602, 2014.     

Choice of Force Field for Proteins Containing Structured and Intrinsically Disordered Regions

Biomolecular force fields optimized for globular proteins fail to properly reproduce properties of intrinsically disordered proteins. In particular, parameters of the water model need to be modified to improve applicability of the force fields to both ordered and disordered proteins. Here, we compared performance of force fields recommended for intrinsically disordered proteins in molecular dynamics simulations of three proteins differing in the content of ordered and disordered regions (two proteins consisting of a well-structured domain and of a disordered region with and without a transient helical motif and one disordered protein containing a region of increased helical propensity). The obtained molecular dynamics trajectories were used to predict measurable parameters, including radii of gyration of the proteins and chemical shifts, residual dipolar couplings, paramagnetic relaxation enhancement, and NMR relaxation data of their individual residues. The predicted quantities were compared with experimental data obtained within this study or published previously. The results showed that the NMR relaxation parameters, rarely used for benchmarking, are particularly sensitive to the choice of force-field parameters, especially those defining the water model. Interestingly, the TIP3P water model, leading to an artificial structural collapse, also resulted in unrealistic relaxation properties. The TIP4P-D water model, combined with three biomolecular force-field parameters for the protein part, significantly improved reliability of the simulations. Additional analysis revealed only one particular force field capable of retaining the transient helical motif observed in NMR experiments. The benchmarking protocol used in our study, being more sensitive to imperfections than the commonly used tests, is well suited to evaluate the performance of newly developed force fields.     

Ordered Disorder of the Astrocytic Dystrophin-Associated Protein Complex in the Norm and Pathology

The abundance and potential functional roles of intrinsically disordered regions in aquaporin-4, Kir4.1, a dystrophin isoforms Dp71, α-1 syntrophin, and α-dystrobrevin; i.e., proteins constituting the functional core of the astrocytic dystrophin-associated protein complex (DAPC), are analyzed by a wealth of computational tools. The correlation between protein intrinsic disorder, single nucleotide polymorphisms (SNPs) and protein function is also studied together with the peculiarities of structural and functional conservation of these proteins. Our study revealed that the DAPC members are typical hybrid proteins that contain both ordered and intrinsically disordered regions. Both ordered and disordered regions are important for the stabilization of this complex. Many disordered binding regions of these five proteins are highly conserved among vertebrates. Conserved eukaryotic linear motifs and molecular recognition features found in the disordered regions of five protein constituting DAPC likely enhance protein-protein interactions that are required for the cellular functions of this complex. Curiously, the disorder-based binding regions are rarely affected by SNPs suggesting that these regions are crucial for the biological functions of their corresponding proteins.     

Conservation of intrinsic disorder in protein domains and families: II. functions of conserved disorder

Regions of conserved disorder prediction (CDP) were found in protein domains from all available InterPro member databases, although with varying frequency. These CDP regions were found in proteins from all kingdoms of life, including viruses. However, eukaryotes had 1 order of magnitude more proteins containing long disordered regions than did archaea and bacteria. Sequence conservation in CDP regions varied, but was on average slightly lower than in regions of conserved order. In some cases, disordered regions evolve faster than ordered regions, in others they evolve slower, and in the rest they evolve at roughly the same rate. A variety of functions were found to be associated with domains containing conserved disorder. The most common were DNA/RNA binding, and protein binding. Many ribosomal proteins also were found to contain conserved disordered regions. Other functions identified included membrane translocation and amino acid storage for germination. Due to limitations of current knowledge as well as the methodology used for this work, it was not determined whether these functions were directly associated with the predicted disordered region. However, the functions associated with conserved disorder in this work are in agreement with the functions found in other studies to correlate to disordered regions. We have established that intrinsic disorder may be more common in bacterial and archaeal proteins than previously thought, but this disorder is likely to be used for different purposes than in eukaryotic proteins, as well as occurring in shorter stretches of protein. Regions of predicted disorder were found to be conserved within a large number of protein families and domains. Although many think of such conserved domains as being ordered, in fact a significant number of them contain regions of disorder that are likely to be crucial to their functions.     

Intrinsic disorder in the Protein Data Bank

The Protein Data Bank (PDB) is the preeminent source of protein structural information. PDB contains over 32,500 experimentally determined 3-D structures solved using X-ray crystallography or nuclear magnetic resonance spectroscopy. Intrinsically disordered regions fail to form a fixed 3-D structure under physiological conditions. In this study, we compare the amino-acid sequences of proteins whose structures are determined by X-ray crystallography with the corresponding sequences from the Swiss-Prot database. The analyzed dataset includes 16,370 structures, which represent 18,101 PDB chains and 5,434 different proteins from 910 different organisms (2,793 eukaryotic, 2,109 bacterial, 288 viral, and 244 archaeal). In this dataset, on average, each Swiss-Prot protein is represented by 7 PDB chains with 76% of the crystallized regions being represented by more than one structure. Intriguingly, the complete sequences of only approximately 7% of proteins are observed in the corresponding PDB structures, and only approximately 25% of the total dataset have >95% of their lengths observed in the corresponding PDB structures. This suggests that the vast majority of PDB proteins is shorter than their corresponding Swiss-Prot sequences and/or contain numerous residues, which are not observed in maps of electron density. To determine the prevalence of disordered regions in PDB, the residues in the Swiss-Prot sequences were grouped into four general categories, "Observed" (which correspond to structured regions), "Not observed" (regions with missing electron density, potentially disordered), "Uncharacterized," and "Ambiguous," depending on their appearance in the corresponding PDB entries. This non-redundant set of residues can be viewed as a 'fragment' or empirical domain database that contains a set of experimentally determined structured regions or domains and a set of experimentally verified disordered regions or domains. We studied the propensities and properties of residues in these four categories and analyzed their relations to the predictions of disorder using several algorithms. "Non-observed," "Ambiguous," and "Uncharacterized" regions were shown to possess the amino acid compositional biases typical of intrinsically disordered proteins. The application of four different disorder predictors (PONDR(R) VL-XT, VL3-BA, VSL1P, and IUPred) revealed that the vast majority of residues in the "Observed" dataset are ordered, and that the "Not observed" regions are mostly disordered. The "Uncharacterized" regions possess some tendency toward order, whereas the predictions for the short "Ambiguous" regions are really ambiguous. Long "Ambiguous" regions (>70 amino acid residues) are mostly predicted to be ordered, suggesting that they are likely to be "wobbly" domains. Overall, we showed that completely ordered proteins are not highly abundant in PDB and many PDB sequences have disordered regions. In fact, in the analyzed dataset approximately 10% of the PDB proteins contain regions of consecutive missing or ambiguous residues longer than 30 amino-acids and approximately 40% of the proteins possess short regions (> or =10 and < 30 amino-acid long) of missing and ambiguous residues.     

Intrinsic Disorder in the Protein Data Bank

Abstract

The Protein Data Bank (PDB) is the preeminent source of protein structural information. PDB contains over 32,500 experimentally determined 3-D structures solved using X-ray crystallography or nuclear magnetic resonance spectroscopy. Intrinsically disordered regions fail to form a fixed 3-D structure under physiological conditions. In this study, we compare the amino-acid sequences of proteins whose structures are determined by X-ray crystallography with the corresponding sequences from the Swiss-Prot database. The analyzed dataset includes 16,370 structures, which represent 18,101 PDB chains and 5,434 different proteins from 910 different organisms (2,793 eukaryotic, 2,109 bacterial, 288 viral, and 244 archaeal). In this dataset, on average, each Swiss-Prot protein is represented by 7 PDB chains with 76% of the crystallized regions being represented by more than one structure. Intriguingly, the complete sequences of only ~7% of proteins are observed in the corresponding PDB structures, and only ~25% of the total dataset have >95% of their lengths observed in the corresponding PDB structures. This suggests that the vast majority of PDB proteins is shorter than their corresponding Swiss-Prot sequences and/or contain numerous residues, which are not observed in maps of electron density. To determine the prevalence of disordered regions in PDB, the residues in the Swiss-Prot sequences were grouped into four general categories, “Observed” (which correspond to structured regions), “Not observed” (regions with missing electron density, potentially disordered), “Uncharacterized,” and “Ambiguous,” depending on their appearance in the corresponding PDB entries. This non-redundant set of residues can be viewed as a ‘fragment’ or empirical domain database that contains a set of experimentally determined structured regions or domains and a set of experimentally verified disordered regions or domains. We studied the propensities and properties of residues in these four categories and analyzed their relations to the predictions of disorder using several algorithms. “Non-observed,” “Ambiguous,” and “Uncharacterized” regions were shown to possess the amino acid compositional biases typical of intrinsically disordered proteins. The application of four different disorder predictors (PONDR® VL-XT, VL3-BA, VSL1P, and IUPred) revealed that the vast majority of residues in the “Observed” dataset are ordered, and that the “Not observed” regions are mostly disordered. The “Uncharacterized” regions possess some tendency toward order, whereas the predictions for the short “Ambiguous” regions are really ambiguous. Long “Ambiguous” regions (>70 amino acid residues) are mostly predicted to be ordered, suggesting that they are likely to be “wobbly” domains.

Overall, we showed that completely ordered proteins are not highly abundant in PDB and many PDB sequences have disordered regions. In fact, in the analyzed dataset ~10% of the PDB proteins contain regions of consecutive missing or ambiguous residues longer than 30 amino-acids and ~40% of the proteins possess short regions (≥10 and <30 amino-acid long) of missing and ambiguous residues.     

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.     

Conservation of intrinsic disorder in protein domains and families: I. A database of conserved predicted disordered regions

Many protein regions have been shown to be intrinsically disordered, lacking unique structure under physiological conditions. These intrinsically disordered regions are not only very common in proteomes, but also crucial to the function of many proteins, especially those involved in signaling, recognition, and regulation. The goal of this work was to identify the prevalence, characteristics, and functions of conserved disordered regions within protein domains and families. A database was created to store the amino acid sequences of nearly one million proteins and their domain matches from the InterPro database, a resource integrating eight different protein family and domain databases. Disorder prediction was performed on these protein sequences. Regions of sequence corresponding to domains were aligned using a multiple sequence alignment tool. From this initial information, regions of conserved predicted disorder were found within the domains. The methodology for this search consisted of finding regions of consecutive positions in the multiple sequence alignments in which a 90% or more of the sequences were predicted to be disordered. This procedure was constrained to find such regions of conserved disorder prediction that were at least 20 amino acids in length. The results of this work included 3,653 regions of conserved disorder prediction, found within 2,898 distinct InterPro entries. Most regions of conserved predicted disorder detected were short, with less than 10% of those found exceeding 30 residues in length.     

More than just tails: intrinsic disorder in histone proteins

Many biologically active proteins are disordered as a whole, or contain long disordered regions. These intrinsically disordered proteins/regions are very common in nature, abundantly found in all organisms, where they carry out important biological functions. The functions of these proteins complement the functional repertoire of "normal" ordered proteins, and many protein functional classes are heavily dependent on intrinsic disorder. Among these disorder-centric functions are interactions with nucleic acids and protein complex assembly. In this study, we present the results of comprehensive bioinformatics analyses of the abundance and roles of intrinsic disorder in 2007 histones from 746 species. We show that all the members of the histone family are intrinsically disordered proteins. Furthermore, intrinsic disorder is not only abundant in histones, but is absolutely necessary for various histone functions, starting from heterodimerization to formation of higher order oligomers, to interactions with DNA and other proteins, and to posttranslational modifications.     

Functional anthology of intrinsic disorder. 2. Cellular components, domains, technical terms, developmental processes, and coding sequence diversities correlated with long disordered regions

Biologically active proteins without stable ordered structure (i.e., intrinsically disordered proteins) are attracting increased attention. Functional repertoires of ordered and disordered proteins are very different, and the ability to differentiate whether a given function is associated with intrinsic disorder or with a well-folded protein is crucial for modern protein science. However, there is a large gap between the number of proteins experimentally confirmed to be disordered and their actual number in nature. As a result, studies of functional properties of confirmed disordered proteins, while helpful in revealing the functional diversity of protein disorder, provide only a limited view. To overcome this problem, a bioinformatics approach for comprehensive study of functional roles of protein disorder was proposed in the first paper of this series (Xie, H.; Vucetic, S.; Iakoucheva, L. M.; Oldfield, C. J.; Dunker, A. K.; Obradovic, Z.; Uversky, V. N. Functional anthology of intrinsic disorder. 1. Biological processes and functions of proteins with long disordered regions. J. Proteome Res. 2007, 5, 1882-1898). Applying this novel approach to Swiss-Prot sequences and functional keywords, we found over 238 and 302 keywords to be strongly positively or negatively correlated, respectively, with long intrinsically disordered regions. This paper describes approximately 90 Swiss-Prot keywords attributed to the cellular components, domains, technical terms, developmental processes, and coding sequence diversities possessing strong positive and negative correlation with long disordered regions.     

Intrinsically disordered protein from a pathogenic mesophile Mycobacterium tuberculosis adopts structured conformation at high temperature

Compared to eukaryotes, the occurrence of "intrinsically disordered" or "natively unfolded" proteins in prokaryotes has not been explored extensively. Here, we report the occurrence of an intrinsically disordered protein from the mesophilic human pathogen Mycobacterium tuberculosis. The Histidine-tagged recombinant Rv3221c biotin-binding protein is intrinsically disordered at ambient and physiological growth temperatures as revealed by circular dichroism and Fourier transform infrared (FTIR) spectroscopic studies. However, an increase in temperature induces a transition from disordered to structured state with a folding temperature of approximately 53 degrees C. Addition of a structure inducing solvent trifluoroethanol (TFE) causes the protein to fold at lower temperatures suggesting that TFE fosters hydrophobic interactions, which drives protein folding. Differential Scanning Calorimetry studies revealed that folding is endothermic and the transition from a disordered to structured state is continuous (higher-order), implying existence of intermediates during folding process. Secondary structure analysis revealed that the protein has propensity to form beta-sheets. This is in conformity with FTIR spectrum that showed an absorption peak at wave number of 1636 cm(-1), indicative of disordered beta-sheet conformation in the native state. These data suggest that although Rv3221c may be disordered under ambient or optimal growth temperature conditions, it has the potential to fold into ordered structure at high temperature driven by increased hydrophobic interactions. In contrast to the generally known behavior of other intrinsically disordered proteins folding at high temperature, Rv3221c does not appear to oligomerize or aggregate as revealed through numerous experiments including Congo red binding, Thioflavin T-binding, turbidity measurements, and examining molar ellipticity as a function of protein concentration. The amino acid composition of Rv3221c reveals that it has 24% charged and 54.9% hydrophobic amino acid residues. In this respect, this protein, although belonging to the class of intrinsically disordered proteins, has distinct features. The intrinsically disordered state and the biotin-binding feature of this protein suggest that it may participate in many biochemical processes requiring biotin as a cofactor and adopt suitable conformations upon binding other folded targets.     

Engineering of an elastic scaffolding polyprotein based on an SH3-binding intrinsically disordered titin PEVK module

Titin is a large elastic protein found in muscle that maintains the elasticity and structural integrity of the sarcomere. The PEVK region of titin is intrinsically disordered, highly elastic and serves as a hub to bind signaling proteins. Systematic investigation of the structure and affinity profile of the PEVK region will provide important information about the functions of titin. Since PEVK is highly heterogeneous due to extensive differential splicing from more than one hundred exons, we engineered and expressed polyproteins that consist of a defined number of identical single exon modules. These customized polyproteins reduce heterogeneity, amplify interactions of less dominant modules, and most importantly, provide tags for atomic force microscopy and allow more readily interpretable data from single-molecule techniques. Expression and purification of recombinant polyprotein with repeat regions presented many technical challenges: recombination events in tandem repeats of identical DNA sequences exacerbated by high GC content, toxicity of polymer plasmid and expressed protein to the bacteria; early truncation of proteins expressed with different numbers of modules; and extreme sensitivity to proteolysis. We have investigated a number of in vitro and in vivo bacterial and yeast expression systems, as well as baculoviral systems as potential solutions to these problems. We successfully expressed and purified in gram quantities a polyprotein derived from human titin exon 172 using Pichia pastoris yeast. This study provides valuable insights into the technical challenges regarding the engineering and purification of a tandem repeat sequence of an intrinsically disordered biopolymer.     

Predicting mostly disordered proteins by using structure-unknown protein data

Background  

Predicting intrinsically disordered proteins is important in structural biology because they are thought to carry out various cellular functions even though they have no stable three-dimensional structure. We know the structures of far more ordered proteins than disordered proteins. The structural distribution of proteins in nature can therefore be inferred to differ from that of proteins whose structures have been determined experimentally. We know many more protein sequences than we do protein structures, and many of the known sequences can be expected to be those of disordered proteins. Thus it would be efficient to use the information of structure-unknown proteins in order to avoid training data sparseness. We propose a novel method for predicting which proteins are mostly disordered by using spectral graph transducer and training with a huge amount of structure-unknown sequences as well as structure-known sequences.     

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

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

DisoMCS: Accurately Predicting Protein Intrinsically Disordered Regions Using a Multi-Class Conservative Score Approach

The precise prediction of protein intrinsically disordered regions, which play a crucial role in biological procedures, is a necessary prerequisite to further the understanding of the principles and mechanisms of protein function. Here, we propose a novel predictor, DisoMCS, which is a more accurate predictor of protein intrinsically disordered regions. The DisoMCS bases on an original multi-class conservative score (MCS) obtained by sequence-order/disorder alignment. Initially, near-disorder regions are defined on fragments located at both the terminus of an ordered region connecting a disordered region. Then the multi-class conservative score is generated by sequence alignment against a known structure database and represented as order, near-disorder and disorder conservative scores. The MCS of each amino acid has three elements: order, near-disorder and disorder profiles. Finally, the MCS is exploited as features to identify disordered regions in sequences. DisoMCS utilizes a non-redundant data set as the training set, MCS and predicted secondary structure as features, and a conditional random field as the classification algorithm. In predicted near-disorder regions a residue is determined as an order or a disorder according to the optimized decision threshold. DisoMCS was evaluated by cross-validation, large-scale prediction, independent tests and CASP (Critical Assessment of Techniques for Protein Structure Prediction) tests. All results confirmed that DisoMCS was very competitive in terms of accuracy of prediction when compared with well-established publicly available disordered region predictors. It also indicated our approach was more accurate when a query has higher homologous with the knowledge database.

Availability

The DisoMCS is available at http://cal.tongji.edu.cn/disorder/.     

Towards an understanding of the role of intrinsic protein disorder on plant adaptation to environmental challenges

Intrinsic protein disorder is an interesting structural feature where fully functional proteins lack a three-dimensional structure in solution. In this work, we estimated the relative content of intrinsic protein disorder in 96 plant proteomes including monocots and eudicots. In this analysis, we found variation in the relative abundance of intrinsic protein disorder among these major clades; the relative level of disorder is higher in monocots than eudicots. In turn, there is an inverse relationship between the degree of intrinsic protein disorder and protein length, with smaller proteins being more disordered. The relative abundance of amino acids depends on intrinsic disorder and also varies among clades. Within the nucleus, intrinsically disordered proteins are more abundant than ordered proteins. Intrinsically disordered proteins are specialized in regulatory functions, nucleic acid binding, RNA processing, and in response to environmental stimuli. The implications of this on plants’ responses to their environment are discussed.     

Markov models of amino acid substitution to study proteins with intrinsically disordered regions

Background

Intrinsically disordered proteins (IDPs) or proteins with disordered regions (IDRs) do not have a well-defined tertiary structure, but perform a multitude of functions, often relying on their native disorder to achieve the binding flexibility through changing to alternative conformations. Intrinsic disorder is frequently found in all three kingdoms of life, and may occur in short stretches or span whole proteins. To date most studies contrasting the differences between ordered and disordered proteins focused on simple summary statistics. Here, we propose an evolutionary approach to study IDPs, and contrast patterns specific to ordered protein regions and the corresponding IDRs.

Results

Two empirical Markov models of amino acid substitutions were estimated, based on a large set of multiple sequence alignments with experimentally verified annotations of disordered regions from the DisProt database of IDPs. We applied new methods to detect differences in Markovian evolution and evolutionary rates between IDRs and the corresponding ordered protein regions. Further, we investigated the distribution of IDPs among functional categories, biochemical pathways and their preponderance to contain tandem repeats.

Conclusions

We find significant differences in the evolution between ordered and disordered regions of proteins. Most importantly we find that disorder promoting amino acids are more conserved in IDRs, indicating that in some cases not only amino acid composition but the specific sequence is important for function. This conjecture is also reinforced by the observation that for of our data set IDRs evolve more slowly than the ordered parts of the proteins, while we still support the common view that IDRs in general evolve more quickly. The improvement in model fit indicates a possible improvement for various types of analyses e.g. de novo disorder prediction using a phylogenetic Hidden Markov Model based on our matrices showed a performance similar to other disorder predictors.